U.S. patent application number 16/789803 was filed with the patent office on 2020-10-08 for stabilizing excipients for therapeutic protein formulations.
The applicant listed for this patent is REFORM BIOLOGICS, LLC. Invention is credited to Daniel G. Greene, Robert P. Mahoney, David S. Soane, Philip Wuthrich.
Application Number | 20200316196 16/789803 |
Document ID | / |
Family ID | 1000004956661 |
Filed Date | 2020-10-08 |
United States Patent
Application |
20200316196 |
Kind Code |
A1 |
Soane; David S. ; et
al. |
October 8, 2020 |
STABILIZING EXCIPIENTS FOR THERAPEUTIC PROTEIN FORMULATIONS
Abstract
The invention encompasses injectable therapeutic formulations
for injection into patients in need thereof, wherein the injectable
formulations are injected using a medical device having a
hydrophobic surface; the injectable therapeutic formulation
comprises a therapeutic protein and a stabilizing excipient that
protects the therapeutic protein from forming particulates or
aggregates in the presence of the hydrophobic surface. Also
included are methods for reducing particulates or aggregates by
using such injectable therapeutic formulations in medical devices
having hydrophobic surfaces.
Inventors: |
Soane; David S.; (Palm
Beach, FL) ; Mahoney; Robert P.; (Newbury, MA)
; Wuthrich; Philip; (Watertown, MA) ; Greene;
Daniel G.; (Reading, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REFORM BIOLOGICS, LLC |
Woburn |
MA |
US |
|
|
Family ID: |
1000004956661 |
Appl. No.: |
16/789803 |
Filed: |
February 13, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US18/46911 |
Aug 17, 2018 |
|
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16789803 |
|
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62547511 |
Aug 18, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/26 20130101;
A61M 2205/0238 20130101; A61K 47/38 20130101; A61K 47/34 20130101;
A61K 39/3955 20130101; A61K 47/183 20130101; A61K 47/12 20130101;
A61K 38/1774 20130101; A61M 5/178 20130101; A61K 47/22
20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 47/34 20060101 A61K047/34; A61K 38/17 20060101
A61K038/17; A61K 47/38 20060101 A61K047/38; A61K 47/22 20060101
A61K047/22; A61K 47/26 20060101 A61K047/26; A61K 47/18 20060101
A61K047/18; A61K 47/12 20060101 A61K047/12; A61M 5/178 20060101
A61M005/178 |
Claims
1. An injectable therapeutic formulation for injection into a
patient in need thereof using a medical device having a hydrophobic
surface, wherein the injectable therapeutic formulation comprises a
therapeutic protein and a stabilizing excipient that protects the
therapeutic protein from forming particulates or aggregates in the
presence of the hydrophobic surface.
2. The injectable therapeutic formulation of claim 1, wherein the
medical device is a syringe.
3. The injectable therapeutic formulation of claim 2, wherein the
syringe is lubricated with silicone oil.
4. The injectable therapeutic formulation of claim 1, wherein the
hydrophobic surface is formed from a silicone-based material.
5. The injectable therapeutic formulation of claim 1, wherein the
hydrophobic surface is coated with a silicone oil.
6. The injectable therapeutic formulation of claim 1, wherein the
therapeutic protein is selected from the group consisting of an
antibody, an antibody-drug conjugate, an enzyme, a cytokine, a
neurotoxin, a fusion protein, an immunogenic protein, a PEGylated
protein, and an antibody fragment.
7-11. (canceled)
12. The injectable therapeutic formulation of claim 1, wherein the
formulation contains at least 100 mg/mL of the therapeutic
protein.
13. (canceled)
14. (canceled)
15. The injectable therapeutic formulation of claim 1, wherein the
formulation contains at least about 1 to about 5000 ppm of the
stabilizing excipient.
16. (canceled)
17. (canceled)
18. The injectable therapeutic formulation of claim 1, wherein the
stabilizing excipient excludes polypropylene glycol block
copolymers.
19. The injectable therapeutic formulation of claim 18, wherein the
stabilizing excipient is selected from the group consisting of
polypropylene glycol, adducts of polypropylene glycol, and random
copolymers comprising propylene oxide units.
20. The injectable therapeutic formulation of claim 19, wherein the
stabilizing excipient is a polypropylene glycol homopolymer.
21-31. (canceled)
32. The injectable therapeutic formulation of claim 1, wherein the
formulation further comprises a second stabilizing excipient.
33-37. (canceled)
38. The injectable therapeutic formulation of claim 1, wherein the
formation of particulates or aggregates is reduced as compared to
that of a control formulation, wherein the control formulation is
identical on a dry weight basis in every way to the therapeutic
formulation except that it lacks the stabilizing excipient.
39. (canceled)
40. A method of reducing the formation of particulates or
aggregates in a medical device having a hydrophobic surface and
containing an injectable therapeutic formulation comprising a
therapeutic protein, wherein the injectable therapeutic formulation
contacts the hydrophobic surface and forms particulates or
aggregates in contact therewith, the method comprising formulating
the injectable therapeutic protein with a stabilizing excipient to
produce the therapeutic formulation for use with the medical
device, wherein the stabilizing excipient is added to the
injectable therapeutic formulation in an amount sufficient to
reduce the formation of particulates or aggregates.
41. The method of claim 40, wherein the hydrophobic surface
comprises silicone.
42. The method of claim 40, wherein the hydrophobic surface is a
surface coated with a silicone oil.
43. The method of claim 40, wherein the therapeutic protein is
selected from the group consisting of an antibody, an antibody-drug
conjugate, an enzyme, a cytokine, a neurotoxin, a fusion protein,
an immunogenic protein, a PEGylated protein, and an antibody
fragment.
44. The method of claim 40, wherein the stabilizing excipient is
selected from the group consisting of polypropylene glycol, adducts
of polypropylene glycol, and random copolymers comprising propylene
oxide units.
45. The method of claim 40, wherein the formation of particulates
or aggregates is reduced as compared to that of a control
formulation, wherein the control formulation is identical on a dry
weight basis in every way to the therapeutic formulation except
that it lacks the stabilizing excipient.
46-52. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of International
Application No. PCT/US18/46911, which designated the United States
and was filed on Aug. 17, 2018, published in English, which claims
the benefit of U.S. Provisional Application No. 62/547,511, filed
Aug. 18, 2017. The entire contents of the above-referenced
applications are incorporated by reference herein.
FIELD OF THE APPLICATION
[0002] This application relates to formulations for stabilizing
therapeutic proteins.
BACKGROUND
[0003] Aqueous formulations of therapeutic proteins (e.g.,
antibodies) are susceptible to degradation through a number of
different mechanisms and as a result of several types of stress
conditions. In general, degradation of a therapeutic protein
formulation occurs when the protein structure is altered slightly
from its fully folded conformation (partial unfolding) exposing
hydrophobic residues that interact with an adjacent protein
molecule in solution forming an irreversible association. Certain
stress conditions such as agitation, freeze/thaw and increased
temperature can induce greater protein unfolding leading to
accelerated aggregation of the protein and degradation of the
protein formulation. Degradation of the protein formulation can be
manifested by protein denaturation, the formation of visible
particles, the formation of aggregates, the formation of subvisible
particles, opalescence of the formulation, loss of biological
activity, loss of percent monomer, loss of yield during production
and purification, and the like. Exposure of the protein formulation
to a liquid/air or liquid/solid interface, such as in agitation or
freeze/thaw conditions, allows for a portion of the protein to
unfold because of the lack of water at the interface to stabilize
the folded structure through hydrogen bonding and hydrophobic
effects. Other mechanisms leading to protein degradation include
oxidation, hydrolysis, proteolysis, photodegradation, and microbial
degradation. It would be desirable to provide a therapeutic protein
formulation with improved stability to make the therapeutic
proteins more resistant to the stress conditions encountered during
their distribution and storage. For example, formulations of
therapeutic proteins can encounter stress conditions like
freeze/thaw cycles, agitation, long term storage, pumping,
filtration, or unrefrigerated storage, where improvements to
stability would be advantageous.
[0004] Protein formulations encounter additional stresses when they
are exposed to the medical devices that are used for administering
these formulations to patients. Syringes, pumps, tubing,
containers, bags, and other medical devices are often used to
store, handle, and administer therapeutic protein formulations.
Contacting a therapeutic formulation with the surfaces of these
devices can result in adsorption of the protein onto the surface of
the device, and this adsorption can lead to a loss of potency or
formation of aggregates or particulates in the therapeutic
formulation. Such aggregates or particulates, forming downstream
from the processing steps used for particle filtration, are not
amenable to removal (whether by filtration or otherwise) before or
during formulation injection.
[0005] In certain examples, syringes can be lubricated with a
silicone oil to minimize the injection force required to administer
the dose to a patient. Silicone oil, however, has been associated
with protein aggregation, even when present in low concentrations.
For example, when solutions of proteins contact silicone
oil-lubricated syringes the protein can become aggregated and form
insoluble particles, as described in Krayukhina et al., J. Pharm.
Sci. 104:527-535 (2015). This adsorption of protein onto silicone
can result in harmful aggregates and particulates, as well as a
reduced potency of the therapeutic protein formulation. Protein
aggregation has also been observed to occur when protein
formulations contact other silicone-based medical device materials
such as seals, gaskets, tubing, bandages, and implants.
[0006] In conventional protein formulations, a small amount of a
nonionic surfactant, typically Polysorbate 80 or Polysorbate 20, is
added to compete with the protein for interfacial surfaces to
reduce protein degradation that occurs with its exposure to such
surfaces. Such nonionic surfactants can also be added to protein
formulations to counteract the adverse effects of contact with
silicones and silicone oils. However, polysorbates themselves can
degrade, either through hydrolysis or oxidation, and the resulting
degradation products promote aggregation and/or reduce solubility
of the protein and destabilize protein formulations. Polysorbates
also pose a problem during the manufacturing process of protein
therapeutics because of their tendency to form micelles. The
formation of micelles can prevent some of the polysorbate from
passing through filters such as during an
ultrafiltration/diafiltration unit operation, causing a
significantly larger polysorbate concentration in the drug
substance than intended. For these reasons, it is desirable to have
a protein formulation that minimizes or is substantially free from
conventional surfactants such as Polysorbate 80 and Polysorbate
20.
SUMMARY
[0007] Disclosed herein, in embodiments, are therapeutic
formulations comprising a protein active ingredient and a
stabilizing excipient. In embodiments, the formulation contains
less than about 1 mg/mL of the protein active ingredient, or
between about 1 .mu.g/mL and about 1 mg/mL of protein active
ingredient, or at least about 1 mg/mL of protein active ingredient,
or at least about 5 mg/mL of protein active ingredient, or at least
100 mg/mL of protein active ingredient, or at least about 200 mg/mL
of protein active ingredient, or at least about 300 mg/mL of
protein active ingredient. In embodiments, the protein active
ingredient is selected from the group consisting of an antibody, an
antibody-drug conjugate, an enzyme, a cytokine, a neurotoxin, a
fusion protein, an immunogenic protein, a PEGylated protein, and an
antibody fragment. In embodiments, the formulation contains at
least about 1 to about 5000 ppm of the stabilizing excipient, or at
least about 1 to about 500 ppm of the stabilizing excipient, or at
least about 10 to about 100 ppm of the stabilizing excipient. In
embodiments, the stabilizing excipient excludes polypropylene block
copolymers.
[0008] In certain embodiments, the stabilizing excipient is
selected from the group consisting of polypropylene glycol
homopolymers, adducts of polypropylene glycol, and random
copolymers comprising propylene oxide units. The stabilizing
excipient can be a polypropylene glycol homopolymer, and it can
have a number-average molecular weight between about 300 and about
5000 Daltons, or a number-average molecular weight of about 425
Daltons, of about 1000 Daltons, or of about 2000 Daltons. In
embodiments, the polypropylene glycol homopolymer is a linear
polymer with at least two hydroxyl groups, which can contain two or
three hydroxyl groups. In embodiments, the polypropylene glycol
homopolymer is a branched polymer, and the branched polymer can be
formed by addition of propylene glycol units to a branched or
multifunctional alcohol or a branched or multifunctional amine. The
branched or multifunctional alcohol can be a sugar, glycerol or
pentaerythritol; the branched or multifunctional amine can be
triethanolamine. In embodiments, the stabilizing excipient is an
adduct of polypropylene glycol. The adduct of polypropylene glycol
can be a reaction product between propylene glycol and an alcohol
or between propylene glycol and an amine. In certain embodiments,
the stabilizing excipient is a hydrophobically modified cellulosic
polymer. The hydrophobically modified cellulosic polymer can be
selected from the group consisting of a methylcellulose, a
hydroxypropyl methylcellulose, a hydroxypropyl cellulose, and a
hydroxyethyl cellulose. In embodiments, the hydrophobically
modified cellulosic polymer is not a sodium carboxymethyl
cellulose. In certain embodiments, the stabilizing excipient is a
polyvinyl alcohol, which can have a molecular weight between about
500 and 500,000 Daltons, and/or which can have a hydrolysis percent
between about 50% and about 100%. In certain embodiments, the
stabilizing excipient is a polyoxazoline, which can be selected
from the group consisting of poly(2-methyl-2-oxazoline),
poly(2-ethyl-2-oxazoline) and poly(2-propyl-2-oxazoline). In
embodiments, the polyoxazoline is poly(2-ethyl-2-oxazoline). In
embodiments, the polyoxazoline has a weight-average molecular
weight between about 1000 and about 500,000 Daltons, or a
weight-average molecular weight between about 5000 and about 50,000
Daltons. In certain embodiments, the stabilizing excipient is
polyvinylpyrrolidone, which can have a molecular weight between
about 1000 and about 1,500,000 Daltons, or a molecular weight
between about 5000 and about 200,000 Daltons, or a molecular weight
between about 10,000 and about 100,000 Daltons.
[0009] In embodiments, the formulations disclosed herein can
further comprises a second stabilizing excipient. In embodiments,
the formulation can exclude conventional surfactants. In other
embodiments, the formulation further comprises between about 1 and
about 5000 ppm of a conventional surfactant, or it comprises
between about 1 and about 100 ppm of the conventional surfactant,
or it comprises between about 10 and about 5000 ppm of the
conventional surfactant, or it comprises between about 100 and
about 2000 ppm of the conventional surfactant. In other
embodiments, the formulation further comprises an additional agent
selected from the group consisting of preservatives, sugars,
polysaccharides, arginine, proline, hyaluronidase, stabilizers, and
buffers.
[0010] Also disclosed herein, in embodiments, are methods of
improving stability in a therapeutic formulation comprising a
protein active ingredient by adding a stability-improving amount of
a stabilizing excipient to the therapeutic formulation. In
embodiments, the stabilizing excipient reduces degradation of the
therapeutic formulation by at least 10%, as compared to a control
formulation lacking the stabilizing excipient, or the stabilizing
excipient reduces degradation of the therapeutic formulation by at
least 30%, as compared to a control formulation lacking the
stabilizing excipient, or the stabilizing excipient reduces
degradation of the therapeutic formulation by at least 50%, as
compared to a control formulation lacking the stabilizing
excipient, or the stabilizing excipient reduces degradation of the
therapeutic formulation by at least 70%, as compared to a control
formulation lacking the stabilizing excipient. Also disclosed
herein, in embodiments, are methods of reducing adverse
infusion-related effects in a patient, comprising administering to
a patient in need thereof a therapeutic formulation comprising a
protein active ingredient and a stabilizing excipient, wherein
infusing the therapeutic formulation into the patient results in
fewer adverse infusion-related effects than infusing a control
formulation into the patient, wherein the control formulation lacks
the stabilizing excipient. In embodiments, the adverse
infusion-related effects are selected from the group consisting of
adverse infusion reactions, adverse immunogenic responses, and
decrease in half-life of a therapeutic protein in the therapeutic
formulation.
[0011] Further disclosed herein, in embodiments, are therapeutic
formulations comprising a therapeutic protein and a stabilizing
excipient, wherein the stabilizing excipient protects the
therapeutic protein from forming particulates or aggregates in the
presence of a hydrophobic surface in a medical device, or in the
presence of a silicone surface in a medical device. In certain
aspects, the silicone surface can be a surface coated with silicone
oil. In embodiments, the medical device can be a syringe.
[0012] The invention also includes a medical device for injection,
for example a syringe, comprising a hydrophobic surface and
comprising an injectable therapeutic formulation, wherein the
therapeutic formulation comprises a therapeutic protein and a
stabilizing excipient that protects the therapeutic protein from
forming particulates or aggregates in the presence of the
hydrophobic surface.
[0013] Also disclosed is a method of reducing the formation of
particulates or aggregates in a medical device having a hydrophobic
surface and containing an injectable therapeutic formulation
comprising a therapeutic protein, wherein the injectable
therapeutic formulation contacts the hydrophobic surface and forms
particulates or aggregates in contact therewith, the method
comprising formulating the injectable therapeutic protein with a
stabilizing excipient to produce the therapeutic formulation for
use with the medical device, wherein the stabilizing excipient is
added to the injectable therapeutic formulation in an amount
sufficient to reduce the formation of particulates or aggregates.
For example, the formation of particulates or aggregates is reduced
as compared to the formation of particulates or aggregates using a
control formulation wherein the wherein the control formulation
lacks the stabilizing excipient.
[0014] In addition, the invention includes a method of reducing
adverse infusion-related effects in a patient, comprising
administering to a patient in need thereof an injectable
therapeutic formulation comprising a protein active ingredient and
a stabilizing excipient using a medical device having a hydrophobic
surface, wherein infusing the injectable therapeutic formulation
into the patient results in fewer adverse infusion-related effects
than infusing a control formulation into the patient, wherein the
control formulation lacks the stabilizing excipient.
BRIEF DESCRIPTION OF FIGURES
[0015] FIG. 1 is a bar graph showing the turbidity of protein
formulations with various excipients.
[0016] FIG. 2 is a bar graph showing the particle counts per mL
using Flocam analysis of protein formulations with various
excipients.
DETAILED DESCRIPTION
[0017] 1. Definitions
[0018] For the purpose of this disclosure, the term "protein"
refers to a sequence of amino acids (i.e., a polypeptide) typically
having a molecular weight between about 1-3000 kiloDaltons (kDa).
Polypeptides with molecular weight of about 1 kDa or higher are
considered to be proteins for the purposes of the invention. In
some embodiments, the molecular weight of the protein is between
about 50-200 kDa; in other embodiments, the molecular weight of the
protein is between about 20-1000 kDa or between about 20-2000 kDa.
As would be understood by skilled artisans, a polypeptide of
sufficient chain length can have a tertiary or quaternary
structure, while shorter polypeptides can lack a tertiary or
quaternary structure. A wide variety of biopolymers are included
within the scope of the term "protein." For example, the term
"protein" can refer to therapeutic or non-therapeutic proteins,
including antibodies, aptamers, fusion proteins, Fc fusion
proteins, PEGylated proteins, synthetic polypeptides, protein
fragments, lipoproteins, enzymes, immunogenic proteins (e.g., as
used in vaccines), structural peptides, peptide drugs, and the
like.
[0019] Those proteins having therapeutic effects may be termed
"therapeutic proteins"; formulations containing therapeutic
proteins in therapeutically effective amounts may be termed
"therapeutic formulations." The therapeutic protein contained in a
therapeutic formulation may also be termed its "protein active
ingredient." Typically, a therapeutic formulation comprises a
therapeutically effective amount of a protein active ingredient and
an excipient, with or without other optional components.
[0020] As used herein, the term "therapeutic" includes both
treatments of existing disorders and preventions of disorders. A
"treatment" includes any measure intended to cure, heal, alleviate,
improve, remedy, or otherwise beneficially affect the disorder,
including preventing or delaying the onset of symptoms and/or
alleviating or ameliorating symptoms of the disorder. The term
"treatment" includes a prophylactic or therapeutic vaccine or other
preventive intervention.
[0021] Those patients in need of a treatment include both those who
already have a specific disorder, and those for whom the prevention
of a disorder is desirable. A disorder is any condition that alters
the homeostatic wellbeing of a mammal, including acute or chronic
diseases, or pathological conditions that predispose the mammal to
an acute or chronic disease. Non-limiting examples of disorders
include cancers, metabolic disorders (e.g., diabetes), allergic
disorders (e.g., asthma), dermatological disorders, cardiovascular
disorders, respiratory disorders, hematological disorders,
musculoskeletal disorders, inflammatory or rheumatological
disorders, autoimmune disorders, gastrointestinal disorders,
urological disorders, sexual and reproductive disorders,
neurological disorders, infectious diseases, and the like.
[0022] The term "mammal" for the purposes of treatment can refer to
any animal classified as a mammal, including humans, domestic
animals, pet animals, farm animals, sporting animals, working
animals, and the like. A "treatment" can therefore include both
veterinary and human treatments. For convenience, the mammal
undergoing such "treatment" can be referred to as a "patient." In
certain embodiments, the patient can be of any age, including fetal
animals in utero.
[0023] In embodiments, a treatment involves providing a
therapeutically effective amount of a therapeutic formulation to a
mammal in need thereof. A "therapeutically effective amount" is at
least the minimum concentration of the therapeutic protein
administered to the mammal in need thereof, to effect a treatment
of an existing disorder or a prevention of an anticipated disorder
(either such treatment or such prevention being a "therapeutic
intervention"). Therapeutically effective amounts of various
therapeutic proteins that may be included as active ingredients in
the therapeutic formulation may be familiar in the art; or, for
therapeutic proteins discovered or applied to therapeutic
interventions hereinafter, the therapeutically effective amount can
be determined by standard techniques carried out by those having
ordinary skill in the art, using no more than routine
experimentation.
[0024] As non-limiting examples, therapeutic proteins can include
mammalian proteins such as hormones and prohormones (e.g., insulin
and proinsulin, synthetic insulin, insulin analogs, glucagon,
calcitonin, thyroid hormones (T3 or T4 or thyroid-stimulating
hormone), parathyroid hormone, gastrin, cholecystokinin, leptin,
follicle-stimulating hormone, oxytocin, vasopressin, atrial
natriuretic peptide, luteinizing hormone, growth hormone, growth
hormone releasing factor, somatostatin, and the like); clotting and
anti-clotting factors (e.g., tissue factor, von Willebrand's
factor, Factor VIIIC, Factor VIII, Factor IX, protein C,
plasminogen activators (urokinase, tissue-type plasminogen
activators), thrombin); cytokines, chemokines, and inflammatory
mediators (e.g., tumor necrosis factor inhibitors); interferons;
colony-stimulating factors; interleukins (e.g., IL-1 through
IL-10); growth factors (e.g., vascular endothelial growth factors,
fibroblast growth factor, platelet-derived growth factor,
transforming growth factor, neurotrophic growth factors,
insulin-like growth factor, and the like); albumins; collagens and
elastins; hematopoietic factors (e.g., erythropoietin,
thrombopoietin, and the like); osteoinductive factors (e.g., bone
morphogenetic protein); receptors (e.g., integrins, cadherins, and
the like); surface membrane proteins; transport proteins;
regulatory proteins; antigenic proteins (e.g., a viral component
that acts as an antigen, as for example in a vaccine). A
therapeutic protein can also be an immunogenic or other protein
(including polypeptide) that is used as a vaccine, where a vaccine
is a natural or synthetic preparation that induces acquired
immunity to a disease. Therapeutic formulations used as vaccines
include toxoid vaccines, protein-based or protein subunit-based
vaccines, or conjugate vaccines. As an illustrative, non-limiting
example, vaccines can contain a surface protein of a virus or a
subunit thereof, as in the HPV virus, the Hepatitis B virus, and
the influenza virus.
[0025] Therapeutic proteins used as vaccines may be derived from
natural sources, for example polypeptides or polypeptide fragments
derived from microorganisms such as fungi (e.g., Aspergillus,
Candida species), bacteria (e.g., Escherichia spp., Staphylococci
spp., Streptococci spp.), protozoa such as sporozoa (e.g.,
Plasmodia), rhizopods (e.g., Entamoeba) and flagellates
(Trypanosoma, Leishmania, Trichomonas, Giardia, etc.), and viruses,
such as (+) RNA viruses, (-) RNA viruses, dsDNA viruses, RNA to DNA
viruses, and DNA to RNA viruses. Examples of viruses from which
vaccines are derived include without limitation Poxviruses (e.g.,
vaccinia), Picornaviruses (e.g., polio), Togaviruses (e.g.,
rubella), Flaviviruses (e.g., HCV); Coronaviruses, Rhabdoviruses
(e.g., VSV); Paramyxovimses (e.g., RSV); Orthomyxovimses (e.g.,
influenza); Bunyaviruses; Arenaviruses, Reoviruses, retroviruses
(e.g., HIV, HTLV); and Hepatitis B virus.
[0026] The term "therapeutic protein" includes without limitation
the full complement of proteins that can be used as drugs, for
example fusion proteins such as etanercept, denileukin diftitox,
alefacept, abatacept, rinolacept, romiplostim,
corifollitropin-alpha, belatacept, aflibercept, ziv-aflibercept,
eftrenonacog-alpha, albiglutide, efraloctocog-alpha, dulaglutide,
and the like.
[0027] The term "therapeutic protein" also includes antibodies. The
term "antibody" is used herein in its broadest sense, to include as
non-limiting examples monoclonal antibodies (including, for
example, full-length antibodies with an immunoglobulin Fc region),
single-chain molecules, bi-specific and multi-specific antibodies,
diabodies, antibody-drug conjugates, antibody compositions having
polyepitopic specificity, and fragments of antibodies (including,
for example, Fab, Fv, Fc, and F(ab')2).
[0028] Antibodies can also be termed "immunoglobulins." An antibody
is understood to be directed against a specific protein or
non-protein "antigen," which is a biologically important material;
the administration of a therapeutically effective amount of an
antibody to a patient can complex with the antigen, thereby
altering its biological properties so that the patient experiences
a therapeutic effect.
[0029] In embodiments, the proteins can be PEGylated, meaning that
they comprise polyethylene glycol) (PEG) and/or polypropylene
glycol) (PPG) units. PEGylated proteins, or PEG-protein conjugates,
have found utility in therapeutic applications due to their
beneficial properties such as improved solubility, improved
pharmacokinetics, improved pharmacodynamics, less immunogenicity,
lower renal clearance, and improved stability. Non-limiting
examples of PEGylated proteins are PEGylated interferons (PEG-IFN),
PEGylated anti-VEGF, PEG protein conjugate drugs, Adagen,
Pegaspargase, Pegfilgrastim, Pegloticase, Pegvisomant, PEGylated
epoetin-.beta., and Certolizumab pegol.
[0030] PEGylated proteins can be synthesized by a variety of
methods such as a reaction of protein with a PEG reagent having one
or more reactive functional groups. The reactive functional groups
on the PEG reagent can form a linkage with the protein at targeted
protein sites such as lysine, histidine, cysteine, and the
N-terminus. Typical PEGylation reagents have reactive functional
groups such as aldehyde, maleimide, or succinimide groups that have
specific reactivity with targeted amino acid residues on proteins.
The PEGylation reagents can have a PEG chain length from about 1 to
about 1000 PEG and/or PPG repeating units. Other methods of
PEGylation include glyco-PEGylation, where the protein is first
glycosylated and then the glycosylated residues are PEGylated in a
second step. Certain PEGylation processes are assisted by enzymes
like sialyltransferase and transglutaminase.
[0031] While the PEGylated proteins can offer therapeutic
advantages over native, non-PEGylated proteins, these materials can
have physical or chemical properties that make them difficult to
purify, dissolve, filter, concentrate, and administer. The
PEGylation of a protein can lead to a higher solution viscosity
compared to the native protein, and this generally requires the
formulation of PEGylated protein solutions at lower
concentrations.
[0032] Those proteins used for non-therapeutic purposes (i.e.,
purposes not involving treatments), such as household, nutrition,
commercial, and industrial applications, may be termed
"non-therapeutic proteins." Formulations containing non-therapeutic
proteins may be termed "non-therapeutic formulations". The
non-therapeutic proteins can be derived from plant sources, animal
sources, or produced from cell cultures; they also can be enzymes
or structural proteins. The non-therapeutic proteins can be used in
household, nutrition, commercial, and industrial applications such
as catalysts, human and animal nutrition, processing aids,
cleaners, and waste treatment.
[0033] An important category of non-therapeutic biopolymers
includes enzymes. Enzymes have a number of non-therapeutic
applications, for example, as catalysts, human and animal
nutritional ingredients, processing aids, cleaners, and waste
treatment agents. Enzyme catalysts are used to accelerate a variety
of chemical reactions. Examples of enzyme catalysts for
non-therapeutic uses include catalases, oxidoreductases,
transferases, hydrolases, lyases, isomerases, and ligases. Human
and animal nutritional uses of enzymes include nutraceuticals,
nutritive sources of protein, chelation or controlled delivery of
micronutrients, digestion aids, and supplements; these can be
derived from amylase, protease, trypsin, lactase, and the like.
Enzymatic processing aids are used to improve the production of
food and beverage products in operations like baking, brewing,
fermenting, juice processing, and winemaking. Examples of these
food and beverage processing aids include amylases, cellulases,
pectinases, glucanases, lipases, and lactases. Enzymes can also be
used in the production of biofuels. Ethanol for biofuels, for
example, can be aided by the enzymatic degradation of biomass
feedstocks such as cellulosic and lignocellulosic materials. The
treatment of such feedstock materials with cellulases and
ligninases transforms the biomass into a substrate that can be
fermented into fuels. In other commercial applications, enzymes are
used as detergents, cleaners, and stain lifting aids for laundry,
dish washing, surface cleaning, and equipment cleaning
applications. Typical enzymes for this purpose include proteases,
cellulases, amylases, and lipases. In addition, non-therapeutic
enzymes are used in a variety of commercial and industrial
processes such as textile softening with cellulases, leather
processing, waste treatment, contaminated sediment treatment, water
treatment, pulp bleaching, and pulp softening and debonding.
Typical enzymes for these purposes are amylases, xylanases,
cellulases, and ligninases.
[0034] Other examples of non-therapeutic biopolymers include
fibrous or structural proteins such as keratins, collagen, gelatin,
elastin, fibroin, actin, tubulin, or the hydrolyzed, degraded, or
derivatized forms thereof. These materials are used in the
preparation and formulation of food ingredients such as gelatin,
ice cream, yogurt, and confections; they area also added to foods
as thickeners, rheology modifiers, mouthfeel improvers, and as a
source of nutritional protein. In the cosmetics and personal care
industry, collagen, elastin, keratin, and hydrolyzed keratin are
widely used as ingredients in skin care and hair care formulations.
Still other examples of non-therapeutic biopolymers are whey
proteins such as beta-lactoglobulin, alpha-lactalbumin, and serum
albumin. These whey proteins are produced in mass scale as a
byproduct from dairy operations and have been used for a variety of
non-therapeutic applications.
[0035] As used herein, the term "conventional surfactant" refers to
an organic surface-active agent capable of lowering the surface
tension between two liquids, or lowering the interfacial tension
between a liquid and a solid. A conventional surfactant is
typically amphiphilic, and can include a hydrophilic "head" and one
or two hydrophobic "tails." The charged character of the head group
allows categorization of the conventional surfactant: a surfactant
with a positively-charged head is termed cationic; a surfactant
with a negatively-charged head is termed anionic; a surfactant with
no charged groups on its head is termed non-ionic; and a surfactant
having a head with two oppositely charged groups is termed
zwitterionic. The tail of the conventional surfactant can comprise
a branched, linear, or aromatic hydrocarbon chain, or it can
comprise a fluorocarbon chain (for fluorosurfactants), or a
siloxane chain (for siloxane surfactants). The hydrophilic
properties of a conventional surfactant can be increased by
including ethoxylated sequences (e.g. polyethylene oxide), while
the lipophilic properties of the conventional surfactant can be
increased by including polypropylene oxide sequences.
[0036] In embodiments, the conventional surfactant can be a
polysorbate, i.e., an emulsifier derived from an ethoxylated
sorbitan ester of a fatty acid. For example, Polysorbate 20
(polyoxyethylene (20) sorbitan monolaurate) and Polysorbate 80
(polyoxyethylene (20) sorbitan monooleate) are commonly used as
conventional surfactants for protein formulations. In other
embodiments, the conventional surfactant can be an ethoxylated
fatty alcohol, a diblock copolymer of ethylene oxide (EO) and
propylene oxide (PO), or a triblock copolymer of EO and PO.
[0037] As used herein, the term "silicone" refers to a chemical
substance comprising silicon-oxygen-silicon bonds, such as
organopolysiloxanes, and mixtures of such substances. "Silicone"
includes any organopolysiloxane, for example silicone oil as may be
used to coat the surfaces of medical devices such as syringes. The
organopolysiloxanes can have a linear or cyclic chemical structure,
and they can be provided in the form of a liquid, a film, a solid,
a grease, an elastomer, or a resin, typically polymers having
(--R.sub.2--Si--O) as a structural unit. A conventional silicone
oil used in medical devices is polydimethylsiloxane such as Dow
Corning 360 Medical Fluid or Dow Corning 365 emulsion.
Organopolysiloxanes and silicone oils are available in different
viscosity specifications, ranging, for example, from about 100 to
about 1,000,000 centistokes (cSt) prior to any curing step, or
about 1000 cSt to about 100,000 cSt, or about 1000 to about 15,000
cSt, or about 12,500 cSt. Exemplary organopolysiloxanes are listed
in U.S. Pat. No. 8,633,034, the entire contents of which are
incorporated herein by reference. The silicone, e.g., a silicone
oil, can be applied as a liquid coating or cured as a siliconized
or crosslinked film onto a surface of a medical device, for
example, a syringe barrel, stopper, needle, tubing, container, bag,
or vial.
[0038] As used herein, the term "medical device" includes those
articles of manufacture that are used in treating a patient. A
medical device can include articles of manufacture such as syringe
assemblies, drug cartridges, metering dispensers for therapeutic
liquids, tubes and valves for therapeutic liquids, catheters,
shunts, vials, needles, needleless injectors, implantable devices,
osmotic pumps, and the like. As a non-limiting example, a medical
device can include a syringe assembly, where such assembly includes
a syringe chamber or barrel adapted for receiving a therapeutic
liquid, a plunger or piston, and a sealing member or stopper. The
chamber can comprise glass, metal, plastic, rubber, or ceramic. A
sealing member is in contact with the chamber to enclose the
therapeutic liquid therein. The sealing member may be formed from
any elastomeric or plastic materials, for example, the synthetic
thermoset elastomers polyisoprene rubber, silicone rubber, and
butyl rubber. In addition, the term "medical device" includes those
articles of manufacture that are used in processing substances
(e.g., compositions and formulations) that are ultimately used in
treating a patient. A medical device, therefore, can include
tubing, valves, injectors, containers, vials, agitators, or other
equipment or surfaces, where such equipment or surfaces contact
substances that are ultimately used in treating a patient.
[0039] 2. General
[0040] The present disclosure relates to aqueous formulations of
therapeutic proteins with stabilizing excipients. As used herein,
the term "stabilizing excipient" refers to an excipient that
reduces the degradation of a therapeutic protein in response to a
stress condition. A stress condition can be any condition that
alters the protein structure, for example by causing greater
protein unfolding, leading to accelerated aggregation and
degradation of the protein formulation. Stress conditions can
include, without limitation, agitation, filtration, freeze/thaw
conditions, lyophilization, exposure to storage temperatures above
5.degree. C., or exposure to a liquid/air or liquid/solid
interface. Other mechanisms involved in stress conditions include
oxidation, hydrolysis, proteolysis, deamidation, disulfide
scrambling, photodegradation, and microbial degradation.
[0041] Stress conditions can also include the contact of the
protein formulation with hydrophobic materials such as plastics,
rubber, resins, lubricants, adhesives, and seals. Under these
circumstances, the protein formulation can undergo adsorption onto
the hydrophobic surface, leading to a loss of potency or the
formation of aggregates or particulates. This situation can also
occur when the protein formulation comes into contact with
silicone-based materials, such as silicone oil lubricated syringes,
elastomeric tubing, and medical devices. Stabilizing excipient
compounds as disclosed herein can protect the protein's
conformational integrity when the protein contacts a hydrophobic
surface or interface, or when the protein contacts a siliconized
surface or a surface bearing silicone oil. Without being bound by
theory, it is thought that the stabilizing excipient compounds
disclosed herein can associate with hydrophobic surfaces or
interfaces, such as silicone oil or siliconized surfaces, thus
preventing, reducing, or minimizing the adsorption of the protein
onto the hydrophobic surfaces.
[0042] It is well known to those skilled in the art of polymer
science and engineering that proteins in solution tend to form
entanglements, which can limit the translational mobility of the
entangled chains and interfere with the protein's therapeutic or
nontherapeutic efficacy. In embodiments, stabilizing excipient
compounds as disclosed herein can suppress protein clustering due
to specific interactions between the excipient compound and the
therapeutic protein in solution.
[0043] In embodiments, the approaches disclosed herein can yield a
liquid formulation having improved stability when compared to a
traditional protein solution. A stable formulation is one in which
the protein contained therein substantially retains its physical
and chemical stability and its therapeutic or nontherapeutic
efficacy upon storage under storage conditions, whether cold
storage conditions, room temperature conditions, or elevated
temperature storage conditions. Advantageously, a stable
formulation can also offer protection against aggregation or
precipitation of the proteins dissolved therein. For example, the
cold storage conditions can entail storage in a refrigerator or
freezer. In some examples, cold storage conditions can entail
conventional refrigerator or freezer storage at a temperature of
10.degree. C. or less. In additional examples, the cold storage
conditions entail storage at a temperature from about 2.degree. to
about 10.degree. C. In other examples, the cold storage conditions
entail storage at a temperature of about 4.degree. C. In additional
examples, the cold storage conditions entail storage at freezing
temperature such as about 0.degree. C. or lower. In another
example, cold storage conditions entail storage at a temperature of
about -30.degree. C. to about 0.degree. C. The room temperature
storage conditions can entail storage at ambient temperatures, for
example, from about 10.degree. C. to about 30.degree. C. Elevated
temperature stability, for example, at temperatures from about
30.degree. C. to about 50.degree. C., can be used as part of an
accelerated aging study to predict the long term storage at typical
ambient (10 to 30.degree. C.) conditions.
[0044] In embodiments, advantageous stabilizing excipients can
comprise polypropylene glycol homopolymers, adducts of
polypropylene glycol, or random copolymers comprising propylene
oxide units. In other embodiments, the stabilizing excipients can
comprise a hydrophobically modified cellulose, which can be a
methylcellulose, a hydroxypropyl methylcellulose, a hydroxypropyl
cellulose, or a hydroxyethyl cellulose, and is not a sodium carboxy
methylcellulose. In other embodiments, the stabilizing excipient is
polyvinyl alcohol. In other embodiments, the stabilizing excipient
is a polyoxazoline, such as poly(2-ethyl-2-oxazoline). In other
embodiments, the stabilizing excipient is polyvinyl
pyrrolidone.
[0045] For example, in embodiments, the stabilizing excipients can
comprise a polypropylene glycol (PPG) homopolymer with a
number-average molecular weight (Mn) between 300 and 5000 Daltons
(Da), such as PPG425, PPG1000, and PPG2000. In embodiments, the
stabilizing excipients can comprise a PPG/PEG copolymer with up to
50% of polyethylene glycol (PEG) repeating units. A PPG excipient
can be a linear polymer with two or three terminal hydroxyl groups.
In embodiments, the stabilizing excipients can comprise a
polypropylene glycol (PPG) adduct, such as a reaction product
between propylene glycol and an alcohol group or an amine group. In
embodiments, the PPG excipient can be in the form of a branched
polymer formed by addition of propylene glycol units to a branched
or multifunctional alcohol or amine like glycerol, triethanolamine,
a sugar, or pentaerythritol.
[0046] In embodiments, the stabilizing excipient can comprise a
hydrophobically modified cellulose such as hydroxypropyl
methylcellulose, methylcellulose, hydroxypropyl cellulose, or
hydroxyethyl cellulose. Low molecular weight hydroxypropyl
methylcellulose (HPMC) and low molecular weight methylcellulose
(MC) are commercially available under the trademark Methocel.RTM.
from Dow Chemical Company (Midland, Mich.). The naming convention
for the Methocel product line is such that the number in the
product name is the viscosity of a 2% solution in water, "LV"
stands for low viscosity, and the first letter indicates the type
(HPMC or MC) and degree of substitution. Low molecular weight HPMC
products such as Methocel E3LV, Methocel E15LV and Methocel K3LV
and low molecular weight MC products (e.g. Methocel A15LV) can be
used as stabilizing excipients.
[0047] In embodiments, the stabilizing excipient can comprise a
polyvinyl alcohol that is prepared from polyvinyl acetate with a
molecular weight between 5000 and 500,000 Da and a degree of
hydrolysis between 50% and 100%. In embodiments, the polyvinyl
alcohol has a degree of hydrolysis from 80% to about 99%, or from
about 83% to about 95%. In embodiments, the polyvinyl alcohol has a
molecular weight between about 10,000 and about 100,000 Da. In
embodiments, the polyvinyl alcohol has a 4% aqueous solution
viscosity at 20-25.degree. C. in the range of about 3 to about 50
cP. In embodiments, the polyvinyl alcohol is a United States
Pharmacopeia (USP) grade.
[0048] In embodiments, the stabilizing excipient can comprise a
polyvinylpyrrolidone (PVP). The PVP excipient can have a molecular
weight of about 1000 to about 1.5 million Da. In embodiments, the
PVP stabilizing excipient can have a molecular weight of about 5000
to about 200,000 Da. In embodiments, the PVP stabilizing excipient
can have a molecular weight of about 10,000 to about 100,000
Da.
[0049] In embodiments, the stabilizing excipient can comprise a
polyoxazoline such as poly(2-methyl-2-oxazoline),
poly(2-ethyl-2-oxazoline) or poly(2-propyl-2-oxazoline). In
embodiments, the stabilizing polyoxazoline excipient can have a
number-average molecular weight of 1,000 to 500,000 Da or 5,000 to
50,000 Da.
[0050] The stabilizing excipient can be added alone, or in
combination with conventional surfactants such as nonionic
surfactants such as Polysorbate 80, Polysorbate 20 and the like.
When a stabilizing excipient is combined with a conventional
surfactant excipient, a lesser amount of conventional excipient may
be required, for example 0 to 100 ppm of the conventional
surfactant, or 100 to 2000 ppm of the conventional surfactant. In
other embodiments, the therapeutic protein formulation contains the
stabilizing excipient and an amount of 10 to 5000 ppm of a
conventional surfactant. In embodiments, the stabilizing excipient
is added to the formulation in amounts ranging from 10 to about
5000 ppm. In embodiments, the stabilizing excipient is added to the
formulation in amounts ranging from 100 to about 1000 ppm. Reducing
the amounts of conventional surfactant in a therapeutic formulation
can offer certain advantages such as improved formulation
stability, improved excipient stability, and reduced foaming
tendency. In embodiments, solutions of therapeutic proteins
containing the stabilizing excipients of the invention can have a
lower foaming tendency compared with solutions of the same
therapeutic proteins without the stabilizing excipients.
[0051] Advantageously, the stabilizing excipients can be selected
so that they do not form micelles in aqueous solution and they can
pass through an ultrafiltration membrane. Advantageously, the
stabilizing excipients can be selected so that they do not increase
the foaming tendency of the formulation. Advantageously, the
stabilizing excipients can be selected so that they do not include
conventional amphiphilic surfactant structures. In embodiments, the
stabilizing excipients can be selected so that they are not
structured as having a hydrophilic head and a hydrophobic tail. In
other embodiments, the stabilizing excipients can be selected so
that they do not comprise block copolymers, for example, so that
block copolymer arrangements such as the (propylene
oxide-co-ethylene oxide) copolymer configurations of PO/EO/PO,
EO/PO or EO/PO/EO are excluded. In embodiments, stabilizing
excipients can be selected that are free of ethylene oxide (EO)
groups, residual ethylene oxide monomer, and/or dioxane byproducts.
In embodiments, the stabilizing excipients are selected so that
they contain no ester linkages. In embodiments, the stabilizing
excipients are purified to minimize the presence of endotoxins or
heavy metals. In embodiments, the stabilizing excipients are USP
grade materials. Stabilizing excipient compounds as disclosed
herein can be natural or synthetic, and, in certain embodiments
they may be substances that the U.S. FDA generally recognizes as
safe (GRAS), or that are well established and commonly used in
registered drug products such as are usually included in
pharmacopoeias, or that are included in a registry or database such
as the FDA's Inactive Ingredient Database
(https://www.accessdata.fda.gov/scripts/cder/iig/).
[0052] 3. Therapeutic Formulations
[0053] In one aspect, the formulations and methods disclosed herein
provide stable liquid formulations, comprising a therapeutic
protein in a therapeutically effective amount and a stabilizing
excipient compound. In embodiments, the formulation can improve the
stability while providing an acceptable concentration of active
ingredients and an acceptable stability. In embodiments, the
formulation provides an improvement in stability when compared to a
control formulation; for the purposes of this disclosure, a control
formulation is a formulation containing the protein active
ingredient that is identical on a dry weight basis in every way to
the therapeutic formulation except that it lacks the excipient
compound. In embodiments, improved stability of the protein
containing formulation is indicated by a lower percentage of
soluble aggregates, a lower percentage of fragments, a decrease in
the number of particulates, a decrease in the number of subvisible
particles, a decrease in the hydrodynamic particle size, or the
suppression of gel formation, as compared to a control formulation
after a stress condition. In embodiments, the stress conditions can
include freeze/thaw cycles, exposure to storage conditions for
>1 month at freezing temperatures (below 0.degree. C.), exposure
to storage conditions for >1 month at refrigerated temperatures
(between 0.degree. C. and 15.degree. C.), exposure to storage
conditions for >1 month at ambient temperatures (between
15.degree. C. and 30.degree. C.), exposure to storage conditions
for >1 week at elevated temperatures (between 30.degree. C. and
100.degree. C.), exposure to agitation stress, exposure to
air/water interfaces, contact with plastic, glass, or metal
surfaces, filtration, column chromatography separation, viral
inactivation, exposure to pH conditions between pH 2 and pH 5,
exposure to pH conditions between pH 8 and pH 12, exposure to
proteolytic enzymes, exposure to lipase enzymes, or exposure to
microbiological contamination.
[0054] It is understood that the stability of a liquid protein
formulation can be affected by a variety of factors, including, but
not limited to: the nature of the protein itself (e.g., enzyme,
antibody, receptor, fusion protein, etc.); its size,
three-dimensional structure, chemical composition, and molecular
weight; its concentration in the formulation; the components of the
formulation besides the protein; the formulation pH range; the
storage conditions for the formulation; and the method of
administering the formulation to the patient. Therapeutic proteins
most suitable for use with the excipient compounds described herein
are preferably essentially pure, i.e., free from contaminating
proteins. In embodiments, an "essentially pure" therapeutic protein
is a protein composition comprising at least 90% by weight of the
therapeutic protein, or preferably at least 95% by weight, or more
preferably, at least 99% by weight, all based on the total weight
of therapeutic proteins and contaminating proteins in the
composition. For the purposes of clarity, a protein added as an
excipient is not intended to be included in this definition. The
therapeutic formulations described herein are intended for use as
pharmaceutical-grade formulations, i.e., formulations intended for
use in treating a mammal, in such a form that the desired
therapeutic efficacy of the protein active ingredient can be
achieved, and without containing components that are toxic to the
mammal to whom the formulation is to be administered.
[0055] In embodiments, the therapeutic formulation contains at
least 1 .mu.g/mL of protein active ingredient. In embodiments, the
therapeutic formulation contains between about 1 .mu.g/mL and about
1 mg/mL of protein active ingredient. In embodiments, the
therapeutic formulation contains at least 1 mg/mL of protein active
ingredient. In embodiments, the therapeutic formulation contains at
least 5 mg/mL of protein active ingredient. In other embodiments,
the therapeutic formulation contains at least 100 mg/mL of protein
active ingredient. In other embodiments, the therapeutic
formulation contains at least 200 mg/mL of protein active
ingredient. In yet other embodiments, the therapeutic formulation
solution contains at least 300 mg/mL of protein active ingredient.
Generally, the excipient compounds disclosed herein are added to
the therapeutic formulation in an amount between about 1 to about
5000 ppm. In embodiments, the excipient compound can be added in an
amount of about 1 to about 500 ppm. In embodiments, the excipient
compound can be added in an amount of about 10 to about 100
ppm.
[0056] In embodiments, the excipient compounds disclosed herein are
added to the therapeutic formulation in a stability-improving
amount. In embodiments, a stability-improving amount is the amount
of an excipient compound that reduces the degradation of the
formulation by at least 10% when compared to a control formulation;
for the purposes of this disclosure, a control formulation is a
formulation containing the protein active ingredient that is
identical on a dry weight basis in every way to the therapeutic
formulation except that it lacks the excipient compound. In
embodiments, the stability-improving amount is the amount of an
excipient compound that reduces the degradation of the formulation
by at least 30% when compared to the control formulation. In
embodiments, the stability-improving amount is the amount of an
excipient compound that reduces the degradation of the formulation
by at least 50% when compared to the control formulation. In
embodiments, the stability-improving amount is the amount of an
excipient compound that reduces the degradation of the formulation
by at least 70% when compared to the control formulation. In
embodiments, the stability-improving amount is the amount of an
excipient compound that reduces the degradation of the formulation
by at least 90% when compared to the control formulation.
[0057] Therapeutic formulations in accordance with this disclosure
have certain advantageous properties. In embodiments, the
therapeutic formulations are resistant to shear degradation, phase
separation, clouding out, precipitation, and denaturing. In
embodiments, the therapeutic formulations are processed, purified,
stored, syringed, dosed, filtered, and centrifuged more
effectively, compared with a control formulation. In embodiments,
the therapeutic formulations can result in fewer adverse
infusion-related effects, for example, adverse infusion reactions,
adverse immunogenic responses, decrease in half-life of a
therapeutic protein in the therapeutic formulation, and the like.
In embodiments, when the therapeutic formulations are administered
to patients, they can experience fewer infusion reactions than
would be experienced with a similar formulation lacking the
stabilizing excipient. In embodiments, when the therapeutic
formulations are administered to patients, they can experience
fewer or less intense immunogenic responses than would be
experienced with a similar formulation lacking the stabilizing
excipient. In embodiments, when the therapeutic formulations are
administered to patients, they can experience less decrease in the
half-life of the therapeutic protein in the body, as compared to a
similar formulation lacking the stabilizing excipient.
[0058] In embodiments, the stabilizing excipient has antioxidant
properties that stabilize the therapeutic protein against oxidative
damage. In embodiments, the therapeutic formulation is stored at
ambient temperatures, or for extended time at refrigerator
conditions without appreciable loss of potency for the therapeutic
protein. In embodiments, the therapeutic formulation is dried down
for storage until it is needed; then it is reconstituted with an
appropriate solvent, e.g., water. Advantageously, the formulations
prepared as described herein can be stable over a prolonged period
of time, from months to years. When exceptionally long periods of
storage are desired, the formulations can be preserved in a freezer
(and later reactivated) without fear of protein denaturation. In
embodiments, formulations can be prepared for long-term storage
that do not require refrigeration. In embodiments, the stabilizing
excipient can be used to improve solubility or stability of protein
therapeutics that have limited water solubility, such as
antibody-drug conjugates.
[0059] In embodiments, the stabilizing excipient provides a
substitute for some or all of the conventional surfactants that are
employed in protein formulations, as described previously. As
described previously, the stabilizing excipient can be added to a
protein formulation alone or in combination with one or more other
excipients, either to replace the conventional surfactant in the
formulation entirely, or to reduce the amount of the conventional
surfactant that is used. In embodiments, the stabilizing excipient
is not an ethoxylated compound, and does not contain residual
amounts of 1,4-dioxane.
[0060] Methods for preparing therapeutic formulations may be
familiar to skilled artisans. The therapeutic formulations of the
present invention can be prepared, for example, by adding the
stabilizing excipient compound to the formulation before or after
the therapeutic protein is added to the solution. The therapeutic
formulation can, for example, be produced by combining the
therapeutic protein and the excipient at a first (lower)
concentration and then processed by filtration or centrifugation to
produce a second (higher) concentration of the therapeutic protein.
Therapeutic formulations can be made with one or more of the
excipient compounds with chaotropes, kosmotropes, hydrotropes, and
salts. Therapeutic formulations can be made with one or more of the
excipient compounds using techniques such as encapsulation,
dispersion, liposome, vesicle formation, and the like. Methods for
preparing therapeutic formulations comprising the stabilizing
excipient compounds disclosed herein can include combinations of
the excipient compounds. Other additives may be introduced into the
therapeutic formulations during their manufacture, including
preservatives, conventional surfactants, sugars, sucrose,
trehalose, polysaccharides, arginine, proline, hyaluronidase,
stabilizers, buffers, and the like. As used herein, a
pharmaceutically acceptable stabilizing excipient compound is one
that is non-toxic and suitable for animal and/or human
administration.
[0061] 4. Protein/Excipient Formulations: Properties and
Processes
[0062] In embodiments, certain of the above-described stabilizing
excipient compounds are used to improve a protein-related process,
such as the manufacture, processing, sterile filling, purification,
and analysis of protein-containing solutions, using processing
methods such as filtration, syringing, transferring, pumping,
mixing, heating or cooling by heat transfer, gas transfer,
centrifugation, chromatography, membrane separation, centrifugal
concentration, tangential flow filtration, radial flow filtration,
axial flow filtration, lyophilization, and gel electrophoresis.
These processes and processing methods can have improved efficiency
due to the improved stability of the proteins in the solution
during manufacture, processing, purification, and analysis steps.
In embodiments, the stabilizing excipient can be added to a
protein-containing solution before a concentration step, and the
stabilizing excipient can improve the efficiency, throughput, or
yield of the concentration step.
[0063] In embodiments, the stabilizing excipients can be added to a
protein formulation to make a more stabilized protein formulation
that resists formation of aggregates and particulates. In
embodiments, the stabilizing excipients can be added to a protein
formulation to make an improved protein formulation that does not
lead to immunogenic responses in patients.
[0064] The stabilizing excipients can be of particular use when
protein formulations are contained in or processed in medical
devices having hydrophobic surfaces, or siliconized surfaces, or
surfaces otherwise treated with a silicone. The stabilizing
excipients can be substituted for conventional surfactants when
formulations are processed in such medical devices, thereby
avoiding the undesirable formation of aggregates and
particulates.
[0065] In embodiments, the stabilizing excipient does not become
concentrated with the protein phase during a filtration-based
concentration step. In embodiments, the stabilizing excipient does
not form micelles when added to a protein-containing solution.
Additionally, equipment-related processes such as the cleanup,
sterilization, and maintenance of protein processing equipment can
be facilitated by the use of the above-identified excipients due to
decreased fouling, decreased denaturing, lower viscosity, and
improved solubility of the protein.
EXAMPLES
[0066] As used herein, the term wt % refers to percentage on a
weight basis.
Example 1: Agitation Stress of ERBITUX.RTM. Formulations
[0067] This example compares the effect of the following
stabilizing excipients in ERBITUX.RTM. formulations that were
subjected to agitation stresses: polypropylene glycol, Mn
.about.425 g/mol (PPG425), polypropylene glycol, Mn 1000 g/mol
(PPG1000), polypropylene glycol, Mn 2000 g/mol (PPG2000),
polyethylene glycol, Mn 1000 g/mol (PEG1000). All stabilizing
excipient reagents were obtained from Sigma-Aldrich, St. Louis,
Mo.
[0068] An ERBITUX.RTM. formulation was prepared as follows. A
commercial cetuximab (ERBITUX.RTM.) drug product distributed in the
U.S. by Eli Lilly & Co. was acquired. According to the FDA drug
label, the commercial formulation contained 2 mg/mL cetuximab, 8.48
mg/mL sodium chloride, 1.88 mg/mL sodium phosphate dibasic
heptahydrate and 0.41 mg/mL sodium phosphate monobasic
monohydrate.
[0069] The ERBITUX.RTM. sample was then reformulated in 15 mM
sodium phosphate and 4.8 mg/mL sodium chloride at pH 7 in the
presence of about 200 ppm of a stabilizing excipient in the
following way. Buffer solutions were prepared by dissolving
approximately 0.1 g sodium phosphate monobasic dihydrate
(Sigma-Aldrich, St. Louis, Mo.), 0.24 g sodium chloride
(Sigma-Aldrich, St. Louis, Mo.) and about 0.1 g of the desired
stabilizing excipient in deionized water, and diluted to a final
mass of about 50 g with additional deionized water. The solution pH
of each buffer was adjusted to about 7 with the dropwise addition
of either 5 M sodium hydroxide or 1 M sodium hydroxide. Buffers
were filtered through 0.2 micron sterile polyethersulfone syringe
filter (GE Healthcare Biosciences, Pittsburgh, Pa.), and 0.4 mL of
each buffer added to sterile 5 mL polypropylene tubes along with
about 3.4 mL of the same buffer containing no excipient. In this
way, a final excipient concentration of about 200 ppm was achieved
in each sample. Amicon Ultra 15 centrifugal concentrator tubes with
a 30 kDa nominal molecular weight cut-off (EMD-Millipore,
Billerica, Mass.) were rinsed with deionized water, filled with 13
mL of ERBITUX.RTM. sample, and centrifuged in a Sorvall Legend RT
centrifuge for about 25 minutes at about 3200.times.g and
25.degree. C. to a final retentate volume of about 1 mL or a
concentration of about 30 mg/mL cetuximab. The filtrate was then
removed and about 0.26 mL was added to each buffer containing 5 mL
sterile polypropylene tube, filtered through 0.2 micron sterile
syringe filters.
[0070] The resulting cetuximab formulations in the 5 mL
polypropylene tubes, having a concentration of about 2 mg/mL
cetuximab and final volume of about 4 mL, were placed on a Daigger
Scientific (Vernon Hills, Ill.) Labgenius orbital shaker at 275 rpm
for agitation stressing. After about 16 hours and about 40 hours of
continuous shaking at ambient temperature, samples were pulled and
analyzed by optical absorbance in a Thermo Fisher Scientific
Evolution spectrophotometer with a 10 mm path length cuvette, and
by dynamic light scattering (DLS) with a ZetaPlus from Brookhaven
Instruments (Holtsville, N.Y.).
[0071] Absorbance at 350 nm and 550 nm was utilized as a
measurement of turbidity, with higher absorbance indicating more
degradation of the cetuximab after stress, due to the formation of
more insoluble particulates. Absorbance values are reported in
Absorbance Units (AU) from the spectrophotometer measurements. The
results are presented in Table 1 below, showing absorbance values
measured after 0, 16, and 40 hours of agitation.
[0072] Dynamic light scattering (DLS) measurements yielded an
effective diameter in nanometers and were not corrected for slight
differences in viscosity and refractive index of the buffers.
Instead, the DLS measurements were used as a more sensitive way
than turbidity for monitoring protein aggregation. The DLS results
are summarized in Table 2.
TABLE-US-00001 TABLE 1 Stabilizing Absorbance at 350 nm (AU)
Absorbance at 550 nm (AU) excipient 0 hrs 16 hrs 40 hrs 0 hrs 16
hrs 40 hrs PEG1000 -0.01 0.00 0.01 -0.02 -0.01 -0.01 PPG425 -0.01
-0.03 -0.04 -0.02 -0.03 -0.04 PPG1000 -0.03 -0.02 0.00 -0.03 -0.02
0.00 PPG2000 -0.04 -0.03 -0.01 -0.04 -0.03 -0.01 None -0.01 0.91
1.62 -0.01 0.52 0.96
TABLE-US-00002 TABLE 2 DLS effective diameter (nm) Stabilizing
excipient 0 hrs 16 hrs 40 hrs PEG1000 11.6 14.2 2900 PPG425 11.8
12.0 11.8 PPG1000 11.8 11.5 11.7 PPG2000 11.6 12.2 12.1 None 11.6
1729 1248
[0073] The four formulations containing a stabilizing excipient all
performed substantially better than the control formulation (which
used the same buffer with no excipient). There was no significant
difference in light absorbance measurements for the four
formulations containing excipient. However, DLS measurements of
effective particle diameter indicated aggregate formation in the
sample containing PEG1000, while the samples containing PPG did not
show any signs of aggregation, regardless of molecular weight
within the range tested in this example. Therefore, it can be
concluded that the various PPG excipients are more effective in
preventing degradation of protein solutions due to agitation and/or
exposure to air/liquid interfaces than PEG1000.
Example 2: FlowCAM Particle Analysis of Stressed Cetuximab
Formulations
[0074] Samples containing about 2 mg/mL cetuximab in phosphate
buffer at pH 7 with 200 ppm excipient, prepared in accordance with
Example 1, were analyzed for insoluble particles by dynamic flow
imaging with a FlowCam VS1 (Fluid Imaging Technologies,
Scarborough, Me.). The FlowCam was equipped with a 20.times.
objective lens and a 50 micron depth flow cell, and operated at a
flow rate of 0.03 mL/min. Measurements were made in triplicate
using a sample volume of 0.2 mL per run. Particles were counted and
grouped into four categories by equivalent spherical diameter using
the VisualSpreadsheet particle analysis software provided with the
FlowCam instrument, and each particle class averaged for the three
sample runs. The results of the experiment are presented in Table 3
below. Particle analysis by FlowCam further demonstrated the
greater stabilization achieved with PPG excipients than the
PEG1000. Within the PPG samples, PPG1000 and PPG2000 performed
similarly and yielded slightly lower particle counts than the
sample with PPG425 as the excipient.
TABLE-US-00003 TABLE 3 Average number of particles per mL after 40
hours shaking: Stabilizing 2-10 .mu.m particles 10-20 .mu.m
particles 20-50 .mu.m particles >50 .mu.m particles excipient
Avg. St.Dev. Avg. St.Dev. Avg. St.Dev. Avg. St.Dev. PEG1000 129445
14028 18474 988 9055 1174 1390 119 PPG425 2119 841 281 139 118 42
22 15 PPG1000 416 103 31 0 0 0 0 0 PPG2000 187 155 21 15 0 0 0
0
Example 3: Evaluation of Low Molecular Weight Hydrophobically
Modified Cellulose
[0075] Low molecular weight hydroxypropyl methylcellulose (HPMC)
and low molecular weight methylcellulose (MC) are hydrophobically
modified cellulose polymers that are commercially available under
the trademark METHOCEL.RTM. from Dow Chemical Company (Midland,
Mich.). The naming convention for the METHOCEL.RTM. product line is
such that the number in the product name is the viscosity of a 2%
solution in water, "LV" stands for low viscosity, and the first
letter indicates the type (HPMC or MC) and degree of substitution.
Three low molecular weight HPMC products (Methocel E3LV, Methocel
E15LV and Methocel K3LV) and one low molecular weight MC product
(Methocel A15LV) were used as stabilizing excipients in this
Example along with Polysorbate 80 (PS80) and polypropylene glycol
2000 (PPG2000) which were obtained from Sigma-Aldrich, St. Louis,
Mo.
[0076] An ERBITUX.RTM. formulation was prepared as follows. A
commercial cetuximab (ERBITUX.RTM.) drug product distributed in the
U.S. by Eli Lilly & Co. was acquired. According to the FDA drug
label, the commercial formulation contained 2 mg/mL cetuximab, 8.48
mg/mL sodium chloride, 1.88 mg/mL sodium phosphate dibasic
heptahydrate and 0.41 mg/mL sodium phosphate monobasic
monohydrate.
[0077] The ERBITUX.RTM. sample was then reformulated in 15 mM
sodium phosphate and 4.8 mg/mL sodium chloride at pH 7 in the
presence of about 200 ppm of a stabilizing excipient in the
following way. Buffer solutions were prepared by dissolving
approximately 0.1 g sodium phosphate monobasic dihydrate
(Sigma-Aldrich, St. Louis, Mo.), 0.24 g sodium chloride
(Sigma-Aldrich, St. Louis, Mo.) and about 0.1 g of the desired
stabilizing excipient in deionized water, and diluted to a final
mass of about 50 g with additional deionized water. The solution pH
of each buffer was adjusted to about 7 with the dropwise addition
of either 5 M sodium hydroxide or 1 M sodium hydroxide. Buffers
were filtered through 0.2 micron sterile polyethersulfone syringe
filter (GE Healthcare Biosciences, Pittsburgh, Pa.), and 0.4 mL of
each buffer added to sterile 5 mL polypropylene tubes along with
about 3.4 mL of the same buffer containing no excipient. In this
way, a final excipient concentration of about 200 ppm was achieved
in each sample. Amicon Ultra 15 centrifugal concentrator tubes with
a 30 kDa nominal molecular weight cut-off (EMD-Millipore,
Billerica, Mass.) were rinsed with deionized water, filled with 13
mL of ERBITUX.RTM. sample, and centrifuged in a Sorvall Legend RT
centrifuge for about 27 minutes at about 3200.times.g and
25.degree. C. to a final retentate volume of about 0.6 mL or a
concentration of about 40 mg/mL cetuximab. The filtrate was then
removed and about 0.2 mL was added to each buffer containing 5 mL
sterile polypropylene tube, filtered through 0.2 micron sterile
syringe filters.
[0078] The resulting cetuximab formulations in the 5 mL
polypropylene tubes, having a concentration of about 2 mg/mL
cetuximab and final volume of about 4 mL, were placed on a Daigger
Scientific (Vernon Hills, Ill.) Labgenius orbital shaker at 275
rpm. After about 16 hours and about 40 hours of continuous shaking
at ambient temperature, samples were pulled and analyzed by light
absorbance in a Thermo Fisher Scientific Evolution
spectrophotometer with a 10 mm path length cuvette, and by dynamic
light scattering (DLS) with a ZetaPlus from Brookhaven Instruments
(Holtsville, N.Y.).
[0079] Absorbance at 350 nm and 550 nm was utilized as a
measurement of turbidity, with higher absorbance indicating more
degradation of the cetuximab after stress, due to the formation of
more insoluble particulates. The absorbance results are reported in
Absorbance Units (AU) from the spectrophotometer measurements and
they are summarized in Table 4 below.
[0080] Dynamic light scattering measurements yielded an effective
diameter in nanometers and were not corrected for slight
differences in viscosity and refractive index of the buffers.
Instead, the DLS measurements were used as a more sensitive way
than turbidity for monitoring protein aggregation. The dynamic
light scattering results are summarized in Table 5 below.
TABLE-US-00004 TABLE 4 Absorbance at 350 nm (AU) Absorbance at 550
nm (AU) Stabilizing excipient 0 hrs 16 hrs 40 hrs 0 hrs 16 hrs 40
hrs Methocel K3LV -0.01 0.01 -0.08 -0.01 0.01 -0.09 Methocel E3LV
-0.01 0.10 -0.06 -0.01 0.10 -0.07 Methocel E15LV 0.01 0.00 -0.06
0.00 0.00 -0.06 Methocel A15LV -0.02 -0.01 -0.07 -0.02 -0.01 -0.07
PS80 -0.01 0.00 -0.04 -0.01 -0.01 -0.04 PPG2000 0.01 0.02 -0.04
0.01 0.01 -0.04
TABLE-US-00005 TABLE 5 DLS effective diameter (nm) Stabilizing
excipient 0 hrs 16 hrs 40 hrs Methocel K3LV 12.2 12.1 12.1 Methocel
E3LV 11.6 12.0 12.3 Methocel E15LV 11.8 12.3 11.9 Methocel A15LV
12.2 12.7 11.9 PS80 11.7 12.3 12.3 PPG2000 11.9 12.5 11.6
[0081] Light absorbance and DLS measurements of samples after
exposure to vigorous shear indicate that low molecular weight HPMC
and MC was able to protect cetuximab from degrading to the same
extent as PS80 and PPG2000.
Example 4: FlowCAM Analysis of Stressed Cetuximab Formulations
[0082] Samples containing about 2 mg/mL cetuximab in phosphate
buffer at pH 7 with 200 ppm excipient prepared in accordance with
Example 3 were analyzed for insoluble particles by dynamic flow
imaging using a FlowCam VS1 (Fluid Imaging Technologies,
Scarborough, Me.). The FlowCam was equipped with a 20.times.
objective lens and a 50 micron depth flow cell, and operated at a
flow rate of 0.03 mL/min. Measurements were made in duplicate using
a sample volume of 1.0 mL per run. Particles were counted and
grouped into four categories by equivalent spherical diameter using
the VisualSpreadsheet particle analysis software provided with the
FlowCam instrument, with the reported value for each particle class
being an average from the two sample runs. The results are set
forth in Table 6 below.
TABLE-US-00006 TABLE 6 Average number of particles per mL after 40
hours shaking: Stabilizing 2-10 .mu.m particles 10-20 .mu.m
particles 20-50 .mu.m particles >50 .mu.m particles excipient
Avg. St.Dev. Avg. St.Dev. Avg. St.Dev. Avg. St.Dev. Methocel 2564
640 309 46 113 3 0 0 K3LV Methocel 893 39 46 15 9 3 0 0 E3LV
Methocel 1773 328 481 7 86 18 0 0 E15LV Methocel 2021 93 145 28 31
6 0 0 A15LV PS80 383 32 31 13 6 6 0 0 PPG2000 493 56 15 9 0 0 0
0
Example 5: Testing of PPG and PEG as Stabilizing Excipients
[0083] This example compares the effect of the following additives
as stabilizing excipients: propylene glycol (PG), dipropylene
glycol (DPG), tripropylene glycol (TPG), polypropylene glycol, Mn
.about.425 g/mol (PPG425), polypropylene glycol, Mn .about.725
g/mol (PPG725), polyethylene glycol, Mn .about.400 g/mol (PEG400),
and Polysorbate 80 (PS80).
[0084] An ERBITUX.RTM. formulation was prepared as follows. A
commercial cetuximab (ERBITUX.RTM.) drug product distributed in the
U.S. by Eli Lilly & Co. was acquired. According to the FDA drug
label, the commercial formulation contained 2 mg/mL cetuximab, 8.48
mg/mL sodium chloride, 1.88 mg/mL sodium phosphate dibasic
heptahydrate and 0.41 mg/mL sodium phosphate monobasic
monohydrate.
[0085] The ERBITUX.RTM. sample was then reformulated in 15 mM
sodium phosphate and 4.8 mg/mL sodium chloride at pH 7 in the
presence of about 200 ppm stabilizing excipient in the following
way. Buffer solutions were prepared by dissolving approximately 0.1
g sodium phosphate monobasic dihydrate (Sigma-Aldrich, St. Louis,
Mo.), 0.24 g sodium chloride (Sigma-Aldrich, St. Louis, Mo.) and
about 0.01 g of the desired stabilizing excipient in deionized
water, and diluted to a final mass of about 50 g with additional
deionized water. The solution pH of each buffer was adjusted to
about 7 with the dropwise addition of either 5 M sodium hydroxide
or 1 M sodium hydroxide. Buffers were filtered through 0.2 micron
sterile polyethersulfone syringe filter (GE Healthcare Biosciences,
Pittsburgh, Pa.), and 3.8 mL of each buffer added to sterile 5 mL
polypropylene tubes. Amicon Ultra 15 centrifugal concentrator tubes
with a 30 kDa nominal molecular weight cut-off (EMD-Millipore,
Billerica, Mass.) were rinsed with deionized water, filled with 14
mL of ERBITUX.RTM. sample, and centrifuged in a Sorvall Legend RT
centrifuge for about 25 minutes at about 3200.times.g and
25.degree. C. to a final retentate volume of about 1 mL or a
concentration of about 30 mg/mL cetuximab. The filtrate was then
removed and about 0.27 mL was added to each buffer containing 5 mL
sterile polypropylene tube, filtered through 0.2 micron sterile
syringe filters.
[0086] The resulting cetuximab formulations in the 5 mL
polypropylene tubes, having a concentration of about 2 mg/mL
cetuximab and final volume of about 4 mL, were placed on a Daigger
Scientific (Vernon Hills, Ill.) Labgenius orbital shaker at 275
rpm. After 16 hours and 39 hours of continuous shaking at ambient
temperature, samples were analyzed by light absorbance in a Thermo
Fisher Scientific Evolution spectrophotometer with a 10 mm path
length cuvette, and by dynamic light scattering (DLS) with a
ZetaPlus from Brookhaven Instruments (Holtsville, N.Y.).
[0087] Absorbance at 350 nm and 550 nm was utilized as a
measurement of turbidity, with higher absorbance indicating more
degradation of the product due to the formation of more insoluble
particulates. Absorbance values are reported in Absorbance Units
(AU) from the spectrophotometer measurements. These results are
presented in Table 7.
[0088] Light scattering measurements yielded an effective diameter
in nanometers and Instead, the DLS measurements were used as a more
sensitive way to monitor aggregation of the protein than turbidity.
The DLS results are summarized in Table 8.
TABLE-US-00007 TABLE 7 Absorbance at 350 nm Absorbance at 550 nm
Test Stabilizing (AU) (AU) No. Excipient 0 hrs 16 hrs 39 hrs 0 hrs
16 hrs 39 hrs 5.1 PPG425 -0.006 -0.007 -0.019 -0.002 -0.005 -0.017
5.2 PS80 -0.025 -0.005 -0.013 -0.019 -0.002 -0.016 5.3 PEG400
-0.026 0.072 0.395 -0.019 0.044 0.192 5.4 PG -0.018 0.156 0.53
-0.013 0.089 0.303 5.5 DPG -0.023 0.355 0.671 -0.019 0.192 0.366
5.6 TPG 0.052 0.184 0.594 0.055 0.104 0.328 5.7 PPG725 -0.015
-0.008 -0.034 -0.012 0.005 -0.033
TABLE-US-00008 TABLE 8 Stabilizing DLS Effective Diameter (nm) Test
No. Excipient 0 hrs 16 hrs 39 hrs 5.1 PPG425 11.6 11.8 11.5 5.2
PS80 11.9 11.5 11.7 5.3 PEG400 11.7 4430 2313 5.4 PG 11.4 3942 1697
5.5 DPG 11.6 6182 2077 5.6 TPG 11.4 4691 2463 5.7 PPG725 11.5 11.6
11.7
[0089] Results from light absorbance and DLS particle size
demonstrate that polypropylene glycol was more effective in
preventing aggregation of cetuximab after severe agitation and high
exposure to air/liquid interface than polyethylene glycol of a
similar molecular weight. Polypropylene glycol also demonstrated
better performance than propylene glycol or its dimer or trimer.
Polypropylene glycol and polysorbate 80 had similar absorbance and
DLS results.
Example 6: Testing of Stabilizing Excipients
[0090] This example compares the effect of the following additives
as stabilizing excipients: hydroxypropyl methylcellulose, Mn
.about.10,000 (HPMC), PPG425, acesulfame K, saccharin, leucine, and
sodium propionate (Na Propionate).
[0091] An ERBITUX.RTM. formulation was prepared as follows. A
commercial cetuximab (ERBITUX.RTM.) drug product distributed in the
U.S. by Eli Lilly & Co. was acquired. According to the FDA drug
label, the commercial formulation contained 2 mg/mL cetuximab, 8.48
mg/mL sodium chloride, 1.88 mg/mL sodium phosphate dibasic
heptahydrate and 0.41 mg/mL sodium phosphate monobasic
monohydrate.
[0092] The ERBITUX.RTM. sample was then reformulated in 15 mM
sodium phosphate at pH 7 in the presence of various stabilizing
excipients in the following way. Buffer solutions containing
excipients were prepared by dissolving approximately 0.1 g sodium
phosphate monobasic dihydrate (Sigma-Aldrich, St. Louis, Mo.) and
the desired excipients in deionized water and adjusting the pH of
the solution to 7 with the dropwise addition of either 1 M sodium
hydroxide or 1 M hydrochloric acid. Solutions were diluted to a
final volume of 50 mL in a volumetric flask with additional
deionized water. Buffers were filtered through 0.2 micron sterile
polyethersulfone syringe filter (GE Healthcare Biosciences,
Pittsburgh, Pa.), and 3.8 mL of each buffer added to sterile 5 mL
polypropylene tubes. Amicon Ultra 15 centrifugal concentrator tubes
with a 30 kDa nominal molecular weight cut-off (EMD-Millipore,
Billerica, Mass.) were rinsed with deionized water, filled with 14
mL of ERBITUX.RTM. sample, and centrifuged in a Sorvall Legend RT
centrifuge for about 25 minutes at about 3200.times.g and
25.degree. C. to a final retentate volume of about 1 mL or a
concentration of about 30 mg/mL cetuximab. The filtrate was then
removed and about 0.28 mL was added to each buffer containing 5 mL
sterile polypropylene tube, filtered through 0.2 micron sterile
syringe filters.
[0093] The resulting cetuximab formulations in the 5 mL
polypropylene tubes, having a concentration of about 2 mg/mL and
final volume of about 4 mL, were placed on a Daigger Scientific
(Vernon Hills, Ill.) Labgenius orbital shaker at 275 rpm. After 16
hours and 36 hours of continuous shaking at ambient temperature,
samples were analyzed by optical absorbance in a Thermo Fisher
Scientific Evolution spectrophotometer with a 10 mm path length
cuvette and by dynamic light scattering (DLS) with a ZetaPlus from
Brookhaven Instruments (Holtsville, N.Y.).
[0094] Absorbance at 350 nm and 550 nm was utilized as a
measurement of turbidity, with higher absorbance indicating more
degradation of the product after stress, due to the formation of
more insoluble particulates. Absorbance values are reported in
Absorbance Units (AU) from the spectrophotometer measurements. In
some cases, samples were allowed to shake for a total of 128 hours
and absorbance measurements obtained. The results are presented in
Table 9 below.
[0095] Dynamic light scattering (DLS) measurements yielded an
effective diameter in nanometers and were not corrected for slight
differences in viscosity and refractive index of the buffers.
Instead, the DLS measurements were used as a more sensitive way
than turbidity for monitoring protein aggregation. The results are
summarized in Table 10 below.
TABLE-US-00009 TABLE 9 Excipient 1 Excipient 2 Test Conc. Conc. ABS
350 nm (AU) ABS 550 nm (AU) No. Name (g/50 mL) Name (g/50 mL) 0 hrs
16 hrs 36 hrs 0 hrs 16 hrs 36 hrs 6.1 HPMC 0.2 NaCl 0.23 0.00 0.01
0 -0.01 0.00 0.00 6.2 KS 0.75 NaCl 0.23 0.00 0.11 0.15 -0.02 0.06
0.07 6.3 LBA 0.75 NaCl 0.23 0.02 0.04 1.15 -0.03 0.02 0.72 6.4
PPG425 0.42 NaCl 0.23 0.02 0.04 0.01 -0.03 0.04 0.02 6.5 Acesulfame
K 0.75 NaCl 0.23 0.02 0.13 1.01 0.02 0.07 0.59 6.6 Saccharin 0.75
NaCl 0.23 0.00 1.53 2.59 -0.03 0.87 1.88 6.7 Leucine 0.49 NaCl 0.23
0.03 0.22 0.44 -0.03 0.12 0.28 6.8 Na Propionate 0.75 -- -- -0.01
0.37 1.51 -0.02 0.21 0.89 6.9 None -- -- -- -0.02 0.63 1.17 -0.02
0.37 0.67 (ERBITUX commercial sample)
TABLE-US-00010 TABLE 10 DLS Effective Diameter (nm) Test No.
Excipients 0 hrs 16 hrs 36 hrs 128 hrs 6.1 See Table 9 14.2 14.2
15.3 N/A 6.2 See Table 9 11.9 4118 4626 4098 6.3 See Table 9 15.3
1588 7277 N/A 6.4 See Table 9 11.9 12.3 12.1 12.3 6.5 See Table 9
11.5 4176 6121 N/A 6.6 See Table 9 11.9 9192 3303 N/A 6.7 See Table
9 11.9 7363 4674 4326 6.8 See Table 9 12.6 3936 3902 N/A 6.9 See
Table 9 11.5 6055 8840 N/A
[0096] An Agilent 1100 series HPLC with auto-sampler was employed
to conduct size-exclusion chromatography (SEC) analysis of the
above formulations to monitor loss of cetuximab monomer after
exposure to shaking stress. Samples were run on a TSKgel Super
SW3000 4.6 mm.times.30 cm column (Tosoh Bioscience) with an
injection volume of 15 microliters and a flow rate of 0.35 mL/min.
An Agilent 1100 series diode array detector was used to measure
eluate absorbance at 280 nm with a bandwidth of 4 nm. The column
temperature was set to 25.degree. C., and the mobile phase was 0.2
M sodium phosphate, pH 6.8. Change in area of the monomer peak
before and after stress was used to quantify monomer loss. Monomer
peak area after shaking stress was reported as a percentage of the
monomer peak area before stress exposure. These results are
presented in Table 11 below.
TABLE-US-00011 TABLE 11 % monomer retained after shaking Test No.
Excipients 16 hrs 36 hrs 6.1 See Table 9 100% 101% 6.2 See Table 9
98% 99% 6.3 See Table 9 97% 73% 6.4 See Table 9 99% 100% 6.5 See
Table 9 97% 86% 6.6 See Table 9 70% 22% 6.7 See Table 9 N/A N/A 6.8
See Table 9 93% 73% 6.9 See Table 9 89% 81%
[0097] Results from light absorbance, DLS particle size and
size-exclusion HPLC (SE-HPLC) demonstrate that hydroxypropyl methyl
cellulose and polypropylene glycol prevent the aggregation of
cetuximab after severe agitation and high exposure to air/liquid
interface. In the presence of either excipient, formation of
visible particles was prevented, as shown by absorbance data.
Preservation of initial monomer size, shown by DLS particle sizing
data, indicates suppression of soluble aggregate formation. SE-HPLC
corroborates these measurements, demonstrating essentially no loss
of monomer in sample.
[0098] The formulation with potassium sorbate also showed
significant improvement over the commercial ERBITUX.RTM. product in
suppressing turbidity after shaking.
Example 7: Shaker Stress of ERBITUX.RTM. Formulations with
Different Amounts of PPG425
[0099] An ERBITUX.RTM. formulation was prepared as follows. A
commercial cetuximab (ERBITUX.RTM.) drug product distributed in the
U.S. by Eli Lilly & Co. was acquired. According to the FDA drug
label, the commercial formulation contained 2 mg/mL cetuximab, 8.48
mg/mL sodium chloride, 1.88 mg/mL sodium phosphate dibasic
heptahydrate and 0.41 mg/mL sodium phosphate monobasic
monohydrate.
[0100] The ERBITUX.RTM. sample was then reformulated in 15 mM
sodium phosphate and 4.8 mg/mL sodium chloride at pH 7 in the
presence of varying amounts of polypropylene glycol having an
average molecular weight of about 425 g/mol (PPG425) in the
following way. Buffer solutions containing PPG425 were prepared by
dissolving approximately 0.1 g sodium phosphate monobasic dihydrate
(Sigma-Aldrich, St. Louis, Mo.) and the desired excipients in
deionized water and adjusting the pH of the solution to 7 with the
dropwise addition of 1 M sodium hydroxide. Solutions were diluted
to a final volume of 50 mL in a volumetric flask with additional
deionized water. In some cases, a buffer containing no PPG425 was
added to a buffer containing PPG425 in such a way as to obtain a
buffer with a lower PPG425 concentration with the same phosphate
and sodium chloride concentrations at pH about 7. Buffers were
filtered through 0.2 micron sterile polyethersulfone syringe filter
(GE Healthcare Biosciences, Pittsburgh, Pa.), and 3.8 mL of each
buffer added to sterile 5 mL polypropylene tubes. Amicon Ultra 15
centrifugal concentrator tubes with a 30 kDa nominal molecular
weight cut-off (EMD-Millipore, Billerica, Mass.) were rinsed with
deionized water, filled with 14 mL of ERBITUX.RTM. sample, and
centrifuged in a Sorvall Legend RT centrifuge for about 25 minutes
at about 3200.times.g and 25.degree. C. to a final retentate volume
of about 1 mL or a concentration of about 30 mg/mL cetuximab. The
filtrate was then removed and about 0.28 mL was added to each
buffer containing 5 mL sterile polypropylene tube, filtered through
0.2 micron sterile syringe filters.
[0101] The resulting cetuximab formulations in the 5 mL
polypropylene tubes, having a concentration of about 2 mg/mL
cetuximab and final volume of about 4 mL, were placed on a Daigger
Scientific (Vernon Hills, Ill.) Labgenius orbital shaker at 275
rpm. After 23 hours and 54 hours of continuous shaking at ambient
temperature, samples were analyzed by light absorbance in a Thermo
Fisher Scientific Evolution spectrophotometer with a 10 mm path
length cuvette and by dynamic light scattering (DLS) with a
ZetaPlus from Brookhaven Instruments Corp. (Holtsville, N.Y.).
[0102] Absorbance at 350 nm and 550 nm was utilized as a
measurement of turbidity, with higher absorbance indicating more
degradation of the product after stress in the form of more
insoluble particulates. Absorbance values are reported in
Absorbance Units (AU) from the spectrophotometer measurements.
These results are presented in Table 12 below.
[0103] Dynamic light scattering (DLS) measurements yielded an
effective diameter in nanometers and were not corrected for slight
differences in viscosity and refractive index of the buffers.
Instead, the DLS measurements were used as a more sensitive way
than turbidity for monitoring protein aggregation. These results
are summarized in Table 13 below.
TABLE-US-00012 TABLE 12 PPG425 Test Conc ABS 350 nm (AU) ABS 550 nm
(AU) No. (mg/mL) 0 hrs 23 hrs 54 hrs 0 hrs 23 hrs 54 hrs 7.1 0.5
-0.03 -0.04 -0.02 -0.02 -0.03 -0.01 7.2 1 -0.02 -0.04 0.00 -0.01
-0.03 0.00 7.3 2 -0.03 -0.04 0.01 -0.03 -0.02 0.01 7.4 5 -0.03
-0.04 0.04 -0.02 -0.02 0.05
TABLE-US-00013 TABLE 13 PPG425 Conc DLS Effective Diameter (nm)
Test No. (mg/mL) 0 hrs 23 hrs 54 hrs 7.1 0.5 11.3 11.7 11.3 7.2 1
11.6 11.4 11.5 7.3 2 11.6 11.6 11.5 7.4 5 11.6 11.9 11.7
[0104] An Agilent 1100 series HPLC with auto-sampler was employed
to conduct size-exclusion chromatography (SEC) analysis of the
above formulations to monitor loss of cetuximab monomer after
exposure to shaking stress. Samples were run on a TSKgel Super
SW3000 4.6 mm.times.30 cm column (Tosoh Bioscience) with an
injection volume of 15 microliters and a flow rate of 0.35 mL/min.
An Agilent 1100 series diode array detector was used to measure
eluate absorbance at 280 nm with a bandwidth of 4 nm. The column
temperature was set to 25.degree. C., and the mobile phase was 0.2
M sodium phosphate, pH 6.8. Change in area of the monomer peak
before and after stress was used to quantify monomer loss. Monomer
peak area after shaking stress was reported as a percentage of the
monomer peak area before stress exposure. These results are set
forth in Table 14 below.
TABLE-US-00014 TABLE 14 PPG425 Conc. % monomer retained after
shaking stress Formulation (mg/mL) 23 hrs 54 hrs 7.1 0.5 98% 98%
7.2 1 99% 99% 7.3 2 100% 99% 7.4 5 99% 98%
[0105] Results from light absorbance, DLS particle size and
size-exclusion HPLC (SE-HPLC) demonstrate the impact polypropylene
glycol from 0.5 mg/mL to 5 mg/mL had in preventing aggregation of
cetuximab after severe agitation and high exposure to air/liquid
interface. Even in the presence of even low excipient
concentration, formation of visible particles was prevented, as
shown by absorbance data. Preservation of initial monomer size
shown by DLS particle sizing data indicates suppression of soluble
aggregate formation. SE-HPLC corroborates these measurements
demonstrating essentially no loss of monomer in sample.
Example 8: Agitation Stress of Formulations Containing 10 mg/mL
Cetuximab
[0106] This example compares the effect of the following additives
in reducing particle formation in an agitated cetuximab
formulation.
[0107] Materials: [0108] Carboxymethylhydroxypropyl guar, CMHPG
(Sigma-Aldrich, St. Louis) [0109] Maltrin M100 (Grain Processing
Corporation, Muscatine, Iowa) [0110] Polyvinylpyrrolidone, 10 kDa
(Sigma-Aldrich, St. Louis, Mo.) [0111] Poly(2-ethyl-2-oxazoline), 5
kDa (Sigma-Aldrich, St. Louis, Mo.) [0112]
Poly(2-ethyl-2-oxazoline), 50 kDa (Sigma-Aldrich, St. Louis, Mo.)
[0113] Polypropylene glycol 1000 (Sigma-Aldrich, St. Louis, Mo.)
[0114] Pluronic F68 (BASF, Florham Park, N.J.) [0115] Polysorbate
80 (Sigma-Aldrich, St. Louis, Mo.)
[0116] A commercial cetuximab (ERBITUX.RTM.) drug product
distributed in the U.S. by Eli Lilly & Co. was acquired.
According to the FDA drug label, the commercial formulation
contained 2 mg/mL cetuximab, 8.48 mg/mL sodium chloride, 1.88 mg/mL
sodium phosphate dibasic heptahydrate and 0.41 mg/mL sodium
phosphate monobasic monohydrate. The ERBITUX.RTM. sample was
reformulated in 10 mM sodium phosphate and 140 mM sodium chloride
at pH 7 in the presence of about 100 ppm or about 200 ppm of
stabilizing excipient in the following way. A stock buffer solution
was prepared by dissolving 1.4 g sodium phosphate monobasic
monohydrate (Sigma-Aldrich, St. Louis, Mo.), and about 8.2 g sodium
chloride (Sigma-Aldrich, St. Louis, Mo.) in deionized water, and
diluted to a final volume of 1 L with additional deionized water.
The solution pH was adjusted to about 7 with the dropwise addition
of 10 M sodium hydroxide. Stabilizing excipient was dissolved in
the resulting buffer by adding 0.02 g or 0.04 g excipient to 50 mL
of the stock buffer at pH 7. Excipient solutions were filtered
through a 0.2 micron sterile polyethersulfone syringe filter (GE
Healthcare Biosciences, Pittsburgh, Pa.), and 0.25 mL of each
excipient solution added to a sterile 2 mL polypropylene tube along
with stock buffer containing no excipient to achieve a volume of
0.71 mL prior to addition of concentrated cetuximab solution.
[0117] Amicon Ultra 15 centrifugal concentrator tubes with a 30 kDa
nominal molecular weight cut-off (EMD-Millipore, Billerica, Mass.)
were rinsed with deionized water, filled with 10 mL of ERBITUX.RTM.
sample, and centrifuged in a Sorvall Legend RT centrifuge at about
3200.times.g and 23.degree. C. to a final retentate volume of about
0.5 mL. The filtrate was then removed and about 0.29 mL was added
to each sterile polypropylene tube, filtered through 0.2 micron
sterile syringe filters.
[0118] The resulting cetuximab formulations in the 2 mL
polypropylene tubes, having a concentration of about 10 mg/mL
cetuximab and a final volume of about 1 mL, were placed on a
Daigger Scientific (Vernon Hills, Ill.) Labgenius orbital shaker at
275 rpm. Samples were analyzed after 40 hours of continuous shaking
by dynamic flow imaging with a FlowCam VS1 (Fluid Imaging
Technologies, Scarborough, Me.).
[0119] The FlowCam was equipped with a 20.times. objective lens and
a 50 micron depth flow cell, and operated at a flow rate of 0.03
mL/min. Measurements were made using a sample volume of 0.5 mL per
run. Particles were counted and reported in four categories
according to equivalent spherical diameter using the
VisualSpreadsheet particle analysis software included with the
instrument.
TABLE-US-00015 TABLE 15 Excipient FlowCam analysis (particles per
mL) Excipient ID conc. (ppm) 2-10 .mu.m 10-20 .mu.m 20-50 .mu.m
>50 .mu.m Total Polyvinylpyrrolidone, 100 564,764 126,021 5560
99 696,444 10K Polysorbate 80 100 3160 172 49 0 3381 Pluronic F68
100 357 12 0 0 369 Poly(2-ethyl-2-oxazoline), 100 2230 320 0 0 2550
5K Poly(2-ethyl-2-oxazoline), 200 46,040 14,826 6090 431 67,387 50K
PPG1000 100 1771 12 12 0 1795 CMHPG 100 1,418,745 659,587 308,738
14,509 2,401,579 Maltin M100 200 1,024,815 705,887 503,525 102,770
2,336,997 None 0 2,962,530 756,922 131,824 6697 3857973
Example 9: Polyvinyl Alcohol as Stabilizer Against Agitation Stress
in Cetuximab Formulations
[0120] This example compares the effect of the following additives
in reducing particle formation in an agitated cetuximab
formulation.
[0121] Materials [0122] Polyvinyl alcohol, 80% hydrolyzed, 9-10 kDa
(Sigma-Aldrich, St. Louis, Mo.) [0123] Polyvinyl alcohol, 87-89%
hydrolyzed, 146-186 kDa (Sigma-Aldrich, St. Louis, Mo.) [0124]
Polypropylene glycol 1000 (Sigma Aldrich, St. Louis, Mo.)
[0125] A commercial cetuximab (ERBITUX.RTM.) drug product
distributed in the U.S. by Eli Lilly & Co. was acquired.
According to the FDA drug label, the commercial formulation
contained 2 mg/mL cetuximab, 8.48 mg/mL sodium chloride, 1.88 mg/mL
sodium phosphate dibasic heptahydrate and 0.41 mg/mL sodium
phosphate monobasic monohydrate. The ERBITUX.RTM. sample was
reformulated in 10 mM sodium phosphate and about 140 mM sodium
chloride at pH 7 in the presence of about 50 ppm or about 100 ppm
of stabilizing excipient in the following way. Buffer solutions
were prepared by dissolving 0.355 g sodium phosphate monobasic
monohydrate (Sigma-Aldrich, St. Louis, Mo.), and about 2 g sodium
chloride (Sigma-Aldrich, St. Louis, Mo.) in deionized water, and
diluted to a final volume of 250 mL with additional deionized
water. The solution pH was adjusted to about 7 with the dropwise
addition of either 10 M sodium hydroxide. Stabilizing excipient was
dissolved in the resulting buffer by adding 0.02 g excipient to 50
mL of the phosphate buffered saline at pH 7. Excipient solutions
were filtered through 0.2 micron sterile polyethersulfone syringe
filter (GE Healthcare Biosciences, Pittsburgh, Pa.), and either 1.0
mL or 0.5 mL of each excipient solution added to sterile 5 mL
polypropylene tubes along with buffer containing no excipient to
achieve a volume of about 3.8 mL prior to addition of concentrated
cetuximab solution.
[0126] Amicon Ultra 15 centrifugal concentrator tubes with a 30 kDa
nominal molecular weight cut-off (EMD-Millipore, Billerica, Mass.)
were rinsed with deionized water, filled with 8.5 mL of
ERBITUX.RTM. sample, and centrifuged in a Sorvall Legend RT
centrifuge at about 3200.times.g and 23.degree. C. to a final
retentate volume of about 0.5 mL. The filtrate was then removed and
about 0.21 mL was added to each sterile polypropylene tube,
filtered through 0.2 micron sterile syringe filters.
[0127] The resulting cetuximab formulations in the 5 mL
polypropylene tubes, having a concentration of about 2 mg/mL
cetuximab and final volume of about 4 mL, were placed on a Daigger
Scientific (Vernon Hills, Ill.) Labgenius orbital shaker at 275
rpm. After about 40 hours of continuous shaking, samples were
pulled and analyzed by dynamic light scattering with a ZetaPlus
from Brookhaven Instruments Corp. (Holtsville, N.Y.), and by
dynamic flow imaging with a FlowCam VS1 (Fluid Imaging
Technologies, Scarborough, Me.).
TABLE-US-00016 TABLE 16 Excipient DLS effective particle size (nm)
Conc. Final (after Identity (ppm) Initial 40 hrs shaking) Polyvinyl
alcohol, 100 11.4 11.4 80% hydrolyzed Polyvinyl alcohol, 50 11.9
25,202 80% hydrolyzed Polyvinyl alcohol, 100 11.9 12.0 87-89%
hydrolyzed Polyvinyl alcohol, 50 13.2 14.4 87-89% hydrolyzed
PPG1000 100 11.8 12.4 None 0 11.6 6817
[0128] The FlowCam was equipped with a 20.times. objective lens and
a 50 micron depth flow cell, and operated at a flow rate of 0.03
mL/min. Measurements were made using a sample volume of 0.5 mL per
run. Particles were counted and reported in four categories
according to equivalent spherical diameter using the
VisualSpreadsheet particle analysis software included with the
instrument.
TABLE-US-00017 TABLE 17 Excipient FlowCam analysis after 40 hrs
shaking (particles/mL) Identity Conc. (ppm) 2-10 .mu.m 10-20 .mu.m
20-50 .mu.m >50 .mu.m Polyvinyl alcohol, 100 1988 171 55 177 80%
hydrolyzed Polyvinyl alcohol, 50 153,069 22,153 10,960 1980 80%
hydrolyzed Polyvinyl alcohol, 100 335 61 6.1 0 87-89% hydrolyzed
Polyvinyl alcohol, 50 128,607 10,475 4130 775 87-89% hydrolyzed
PPG1000 100 506 30 55 0 None 0 2,936,829 563,874 144,868 6800
[0129] In all cases the presence of the additive significantly
reduced the number of particles generated during the agitation
stress compared to sample with no additive, as demonstrated by the
FlowCam data.
Example 10: The Impact of the Cellulosic Modification on the
Stability of Cetuximab Formulations
[0130] Two modified cellulosic materials, hydroxypropyl cellulose
(HPC), and sodium carboxymethyl cellulose (CMC), were examined for
their ability to stabilize cetuximab solutions to particle
formation. The excipients were purchased from Sigma-Aldrich (St.
Louis, Mo.) and used without further modification. Cetuximab was
purchased under the trade name ERBITUX.RTM. (Eli Lilly,
Indianapolis, Ind.) from Clinigen Group (Yardley, Pa.). The HPC had
a weight-average molecular weight of 80,000 and a number-average
molecular weight of 10,000. The CMC had a weight-average molecular
weight of 90,000. The number-average was not reported for the CMC.
The cetuximab concentration was 2 mg/mL in all experiments. The HPC
and CMC concentrations are listed in Table 18. A control sample
with no stabilizing excipient was also prepared. All solutions were
prepared in phosphate buffered saline, pH 7. Four mL of each sample
was placed into sterile 5 mL polypropylene tubes. The samples were
stressed by shaking on an orbital shaker at 275 rpm for 40 hours.
Particulate formation was quantified by measuring the absorbance at
350 nm and the particle size was measured using dynamic light
scattering (DLS). A Brookhaven ZetaPlus instrument was used to
perform the DLS experiments. The cumulants expansion was fit the
DLS intensity autocorrelation functions to estimate the particle
sizes. DLS measurements were made both prior to and after
shaking.
[0131] Solutions containing HPC are less turbid than the control
(Table 18) and the apparent particle size does not change during
the experiment, indicating that HPC is effective in stabilizing
cetuximab towards particle formation. However, solutions containing
CMC have turbidities and final particle sizes similar to that of
the control, indicating that CMC is not effective in stabilizing
cetuximab towards particle formation. The results here illustrate
that the type of modification is important in the ability of the
cellulosic material to protect against particulate formation in
protein solutions.
TABLE-US-00018 TABLE 18 Cellulosic Absorbance Initial DLS Final DLS
concentration at 350 nm particle size particle size Sample (ppm)
(AU) (nm) (nm) Control N/A 0.749 12.2 10,421 CMC 2151 0.560 14.1
13,936 CMC 203 0.628 11.9 17,620 HPC 1850 0.071 14.0 13.3 HPC 199
0.065 11.9 11.8
Example 11: Excipients that Stabilize Cetuximab Formulations to
Particle Formation
[0132] In this example, cetuximab at 2 mg/mL is stressed in the
presence of several different stabilizing excipients. The
excipients are polyvinyl pyrrolidone (PVP) with weight-average
molecular weight of 40,000, polyvinyl alcohol (PVOH) with
weight-average molecular weight of 13,000-23,000 and 87-89%
hydrolyzed residues, 2-hydroxyethyl cellulose (HEC) with
viscosity-average molecular weight of 90,000, and
poly(2-ethyl-2-oxazoline) (POX5000) with number-average molecular
weight of 5000 and polydispersity less than or equal to 1.2, and
aspartame. The excipients were purchased from Sigma-Aldrich (St.
Louis, Mo.) and used without further modification. Cetuximab was
purchased under the trade name ERBITUX.RTM. (Eli Lilly,
Indianapolis, Ind.) from Clinigen Group (Yardley, Pa.). The
concentrations of each excipient are listed in Table 19. A control
sample was also prepared with no stabilizing excipient. All
experiments were performed in a phosphate buffered saline at pH 7.
Four mL of each sample was put into sterile 5 mL polypropylene
tubes, which were then placed on an orbital shaker set to 275 rpm.
The samples were shaken for 40 hours, after which they were
analyzed for particulate formation by an absorbance measurement at
405 nm to estimate turbidity and the total number of particles were
counted using a FlowCam imaging device. The turbidity measurements
were performed with a BioTek Synergy plate reader and corrected for
the path length of the liquid height in the microplate. The FlowCam
was equipped with a 20.times. objective lens and a 50 .mu.m depth
flow cell, and operated at a flow rate of 0.03 mL/min. Measurements
were made using a sample volume of 0.5 mL per run. The control
sample was run through the FlowCam last and partially clogged the
flow cell, which prevented an accurate particle count. This makes
the number listed in Table 19 a lower bound on the total number of
particles. At the use levels indicated in Table 19, all of the
additives stabilize cetuximab to particulate formation as indicated
by a reduction in the absorbance at 405 nm and a reduction in the
total number of particles per mL.
TABLE-US-00019 TABLE 19 % Number % Excipient Absorbance Reduction
of Reduction concentration at 405 nm of ABS405 particles in
particles Sample (ppm) (AU) nm per mL per mL Control N/A 0.340 0
>1,184,884 0 PVP 1801 0.044 87 14,868 >98.7 PVOH 197 0.032 90
898 >99.9 HEC 199 0.041 88 7559 >99.4 POX5000 197 0.040 88
15,112 >98.7 Aspartame 5886 0.066 81 423,636 >64.2
Example 12: The Foaming Propensity of Stabilizing Excipients
[0133] Solutions of concentration 1000 ppm w/v of poly(propylene
glycol) with M.sub.n 1000 (PPG1000, Sigma Aldrich, St. Louis, Mo.),
poly(2-ethyl-2-oxazoline) with M.sub.n 5000 and PDI.ltoreq.1.2
(POX5000, Sigma Aldrich, St. Louis, Mo.), Methocel E3LV (Dow
Chemical Company, Midland, Mich.), polysorbate 80 (PS80, Sigma
Aldrich, St. Louis, Mo.), and Pluronic F68 (F68, BASF Corporation)
were prepared using ultrapure deionized water (18.2 M.OMEGA.cm at
25.degree. C.). 3 mL of each solution was placed into a 5-mL
polypropylene tube and vortexed on a Mini Vortex Mixer (VWR,
Radnor, Pa.) on mixer setting #8 for 15 seconds. The foam heights
were measured after vortexing and are listed in Table 20. The times
for the foams to dissipate were also recorded and are listed in
Table 20. The foams for the POX5000 and F68 samples did not
dissipate over the course of the experiment. The stopping points
are listed in Table 20.
TABLE-US-00020 TABLE 20 Excipient Foam height (in) Foam dissipation
time (s) PPG1000 0.25 4 POX5000 0.63 27 Methocel E3LV 1.00 >1125
PS80 0.25 7 F68 1.25 >1125
Example 13: The Recovery of Stabilizing Excipients after
Ultrafiltration
[0134] Solutions of concentration 1000 ppm w/v of poly(propylene
glycol) with M.sub.n 1000 (PPG1000, Sigma Aldrich, St. Louis, Mo.),
poly(2-ethyl-2-oxazoline) with M.sub.n 5000 and PDI.ltoreq.1.2
(POX5000, Sigma Aldrich, St. Louis, Mo.), Methocel E3LV (Dow
Chemical Company, Midland, Mich.), polysorbate 80 (PS80, Sigma
Aldrich, St. Louis, Mo.), and Pluronic F68 (F68, BASF Corporation)
were prepared using ultrapure deionized water (18.2 M.OMEGA.cm at
25.degree. C.).
[0135] The filter devices used are Amicon Ultra-4 centrifugal
devices with 30,000 molecular weight cut-off (EMD Millipore). A
Sorvall Legend RT Centrifuge was used to filter the feed volume
through the membrane at 4,000 rpm for 10 minutes at 25.degree. C.
An Agilent 1100 HPLC system with attached refractive index detector
(RID) was used to measure the concentration of the material in
feed, retentate and filtrate volumes.
[0136] A standard curve was prepared initially for each solution.
Concentrations of 1000, 700, 500, 400, 200 and 100 ppm were
prepared and analyzed on HPLC-RID to determine peak area. The peak
area was then plotted against concentration and a linear response
was observed for all solutions at a range from 100-1000 ppm.
[0137] The filter devices were pre-rinsed by addition of 4 mL of DI
water and centrifugation for 10 minutes at 4,000 rpm and 25.degree.
C. The filtered rinse water was then discarded before the addition
of 4 mL of 1000 ppm excipient solution. The samples were then
centrifuged using conditions as stated above. The filtrate from
filter devices was emptied into clean 30 mL tubes (tare weighed)
after centrifugation. The process was repeated four additional
times for a total of 20 mL filtrate material. The total amount of
filtrate was determined by mass. Each condition was performed in
triplicate.
[0138] A volume of 100 .mu.L of stock, retentate, and filtrate was
removed and 20 .mu.L injections were prepared to determine peak
area by refractive index detection (RID). Using the standard curve
formula, the concentrations were determined by inputting the peak
areas. The amount of excipient recovered in the filtrate and
retentate was determined by multiplying the total filtrate and
retentate volumes by the measured concentrations. The mass of
excipient recovered in the filtrate was compared to the initial
mass added to filtration device to determine a percent
recovery.
[0139] The percent recoveries of the excipients studied here are
given in Table 21. The recoveries of PPG1000, Methocel E3LV,
POX5000, and Pluronic F68 are greater than those of PS80.
TABLE-US-00021 TABLE 21 Mass Mass Mass excipient excipient Average
excipient in recovered Percent percentage added retentate in
filtrate recovered recovered Excipient (mg) (mg) (mg) in filtrate
in filtrate PS80 20.1 10.4 8.72 43.4% 40.5% 20.1 10.2 7.84 39.0%
20.1 10.0 7.87 39.1% PPG1000 20.2 0.35 20.7 102% 103% 20.2 0.35
20.6 102% 20.2 0.35 21.2 105% POX5000 20.2 0.45 19.9 98.4% 100%
20.2 0.45 20.4 101% 20.2 0.44 20.3 101% Methocel 16.7 4.71 13.8
82.6% 79.6% E3LV 16.7 4.12 12.9 77.4% 16.7 4.86 13.1 78.9% F68 23.8
0.81 21.55 90.6% 90.7% 23.8 0.81 21.62 90.8% 23.8 0.84 21.58
90.7%
Example 14: Excipients that Stabilize Abatacept
[0140] Abatacept was purchased under the trade name ORENCIA.RTM.
(Bristol-Meyers Squibb, Princeton, N.J.) from the Clinigen Group
(Yardley, Pa.). ORENCIA.RTM. was reconstituted to 25 mg/mL
abatacept in ultrapure deionized water with resistivity of 18.2
M.OMEGA.cm (EMD Millipore, Billerica, Mass.) as per the package
insert instructions. A 14-mM monosodium phosphate buffer (Sigma
Aldrich, St. Louis, Mo.), pH 7.5 with 25 mM NaCl (Sigma Aldrich,
St. Louis, Mo.) was prepared. Stock solutions of concentration 2000
ppm by weight of poly(propylene glycol) with M.sub.n 1000 (PPG1000,
Sigma Aldrich, St. Louis, Mo.), poly(2-ethyl-2-oxazoline) with
M.sub.n 5000 and PDI.ltoreq.1.2 (POX5000, Sigma Aldrich, St. Louis,
Mo.), and Methocel E3LV (Dow Chemical Company, Midland, Mich.) were
prepared using the previously described buffer. Samples were
prepared by mixing the reconstituted ORENCIA.RTM. with the
excipient stock solution and the buffer to a final protein
concentration of 2.5 mg/mL, final excipient concentrations of
approximately 1800 ppm and 200 ppm. 1 mL of each sample was placed
into a sterile 2 mL polypropylene tube. The tubes were shaken for
18 hours at 25.degree. C. on a Multi-Therm Shaker (Southwest
Science, Roebling, N.J.). An unstressed control sample with no
excipient was also included (Sample 1, Table 22). The samples were
assayed for apparent particle size by dynamic light scattering
(DLS) and total particle count by FlowCam imaging. Forty .mu.L of
each sample was loaded into a 384-well microplate (Aurora
Microplates, Whitefish, Mont.). Air bubbles were removed from the
microplate by centrifuging the plate at 400.times.g for 1 minute.
The plate was then assayed for apparent particle size by DLS
(DynaPro Plate Reader II, Wyatt, Santa Barbara, Calif.). The
instrument control and data fitting were performed using the
DYNAMICS software package (Wyatt, Santa Barbara, Calif.). The
incident wavelength was 830 nm and the scattering angle was
158.degree.. The intensity autocorrelation functions were generated
using five 5-second exposures and were fit to the cumulants
expansion to estimate the particle diffusivity. The apparent
hydrodynamic radii (Rh) were calculated from the diffusivities via
the Stokes-Einstein relation. Sub-visible particle (greater than 2
microns in size) formation was quantified using a FlowCam VS1
analyzer (Fluid Imaging Technologies, Scarborough, Me.). The
FlowCam was equipped with a 20.times. objective lens and a
50-micron depth flow cell, and operated at a flow rate of 0.03
mL/min. 0.5 mL of each sample was assayed for particle counts.
[0141] All of the excipients in this example stabilize abatacept
formulations to a shaking stress as indicated by a decrease in the
particle count and hydrodynamic size as compared to the control
(Sample 2, Table 22).
TABLE-US-00022 TABLE 22 Excipient concentration Stressed? R.sub.h
Particles/ Sample Excipient (ppm) (Yes/No) (nm) mL 1 None N/A No
5.3 571 2 None N/A Yes 65.6 292,413 3 PPG1000 1800 Yes 5.0 3591 4
POX5000 1750 Yes 7.8 1021 5 Methocel 2020 Yes 5.3 2595 E3LV 6
PPG1000 200 Yes 5.1 4550 7 POX5000 195 Yes 6.2 69,474 8 Methocel
225 Yes 5.1 2813 E3LV
Example 15: PPG1000 Ultrafiltration without Concentration
[0142] Poly(propylene glycol) 1000 (PPG1000), purchased from
Sigma-Aldrich, was prepared at a concentration of 1000 ppm (0.1%)
in ultrapure deionized (DI) water (18.2 M.OMEGA.cm). Polysorbate 80
(PS80), purchased from Sigma-Aldrich, was prepared at a
concentration of 1000 ppm (0.1%) in ultrapure deionized (DI) water
(18.2 M.OMEGA.cm). The PPG1000 and PS80 solutions were processed by
ultrafiltration in the following way. Amicon Ultra-4 centrifugal
filter devices with a 30,000 molecular weight cut-off (EMD
Millipore) were pre-rinsed with 5 mL of DI water. A Sorvall Legend
RT Centrifuge was used to spin down the DI water to wash filter
membrane at 4000 rpm for 10 minutes at 24.degree. C. A total of 3
washes were completed for a total wash volume of 15 mL of DI water
before samples were placed in the filter devices. After rinsing the
filter devices, 5 mL of PPG1000 or PS80 at 0.1% was added and the
devices were centrifuged at 4000 rpm for 5 minutes at 24.degree. C.
A volume of 100 .mu.L of retentate and filtrate were removed from
the filtration devices for analysis using a refractive index
detector (RID) in line with an Agilent 1100 HPLC system.
[0143] The relative concentrations of PPG1000 and PS80 in the
starting solution, the retentate, and the filtrate were measured
with the HPLC/RID system. The retentate and filtrate peak areas
listed in Table 23 for PPG1000 are nearly identical, indicating
that PPG1000 does not concentrate in the retentate during
ultrafiltration. In the same ultrafiltration conditions, PS80
concentrates in the retentate by a factor of 90 compared to the
initial stock concentration and is depleted in the filtrate by a
factor of 20 less than the initial stock concentration as shown by
the peak areas recorded in Table 23.
TABLE-US-00023 TABLE 23 Stock Test Solution Retentate Filtrate No.
Stock solution (peak area) (peak area) (peak area) 1 0.1% PS80
40,794 3,617,387 2,109 2 0.1% PS80 40,794 3,623,979 2,330 3 0.1%
PS80 40,794 2,940,782 [no detectable PS80] 4 0.1% PPG1000 162,226
166,405 165,743 5 0.1% PPG1000 162,226 164,225 165,307 6 0.1%
PPG1000 162,226 163,976 167,556
Example 16: Stabilization of Cetuximab Formulation in Presence of
Silicone Oil
[0144] A commercial cetuximab (ERBITUX.RTM.) drug product
distributed in the U.S. by Eli Lilly & Co. was acquired.
According to the FDA drug label, the commercial formulation
contained 2 mg/mL cetuximab, 8.48 mg/mL sodium chloride, 1.88 mg/mL
sodium phosphate dibasic heptahydrate and 0.41 mg/mL sodium
phosphate monobasic monohydrate. The stock cetuximab formulation at
2 mg/mL was centrifugally concentrated in an Amicon Ultra 15 device
having a 30 kDa molecular weight cut-off (EMD Millipore, Billerica,
Mass.). The resulting cetuximab formulation had a concentration of
4.1 mg/mL as measured by absorbance at 280 nm using an extinction
coefficient of 1.4 mL/mg-cm, and was used to prepare test samples
below.
[0145] Phosphate buffered saline (PBS) was prepared following the
formulation recipe described on the ERBITUX.RTM. drug label to
achieve a 10 mM sodium phosphate, 145 mM sodium chloride solution
at pH 7.2. Stock solutions of excipient of concentration 1000 ppm
w/w of poly(propylene glycol) with Mn 1000 (PPG1000, Sigma Aldrich,
St. Louis, Mo.), poly(propylene glycol) with Mn 425 (PPG425, Sigma
Aldrich, St. Louis, Mo.), poly(2-ethyl-2-oxazoline) with Mn 5000
and PDI 10.ltoreq.1.2 (POX5000, Sigma Aldrich, St. Louis, Mo.),
polysorbate 80 (PS80, Sigma Aldrich, St. Louis, Mo.), and Pluronic
F68 (F68, BASF Corporation) were prepared by dissolving 0.03 g of
excipient in 30 g of PBS. All stock excipient solutions in PBS were
filtered through Whatman 0.2 micron polyethersulfone syringe
filters prior to use.
[0146] Formulations containing cetuximab at 2 mg/mL in the presence
of 200 ppm excipient were prepared by combining 317 .mu.L of PBS
with 200 .mu.L of stock excipient solution and 483 .mu.L stock
cetuximab solution in a sterile 2 mL polypropylene microcentrifuge
tube. Each excipient condition was prepared in triplicate. To each
tube was then added 10 .mu.L of silicone oil having a viscosity of
1000 centistokes (378399, Sigma-Aldrich, St. Louis, Mo.). Sample
tubes were then fastened horizontally on an orbital shaker
(Heathrow Scientific, Vernon Hills, Ill.) and agitated continuously
for 40 hours at 275 rpm and ambient temperature (20-25.degree. C.).
After exposure to this agitation stress, 125 .mu.L of each sample
was loaded onto a clear 96 half-well microplate (Greiner Bio-One,
Monroe, N.C.) and absorbance at 350 nm measured with a BioTek
Synergy HT plate reader (BioTek, Winooski, Vt.). The results of
these tests are shown in Table 24 below, where a higher absorbance
at 350 nm indicates higher turbidity and greater number of
particles, and selected results are illustrated in FIG. 1.
TABLE-US-00024 TABLE 24 Absorbance Excipient Excipient conc.
Agitation at 350 nm Test No. added (ppm) (hrs) (a.u.) 1 F68 200 40
0.932 2 F68 200 40 0.867 3 F68 200 40 0.954 4 PS80 200 40 0.098 5
PS80 200 40 0.078 6 PS80 200 40 0.064 7 PPG425 200 40 1.87 8 PPG425
200 40 2.00 9 PPG425 200 40 1.92 10 PPG1000 200 40 0.056 11 PPG1000
200 40 0.057 12 PPG1000 200 40 0.055 13 POX5000 200 40 2.13 14
POX5000 200 40 2.14 15 POX5000 200 40 2.05 16 None 0 40 1.91 17
None 0 40 1.79 18 None 0 40 1.67 19 None 0 0 0.037 20 None 0 0
0.039 21 None 0 0 0.037
Example 16: FlowCam Analysis of Cetuximab Solutions Stressed in the
Presence of Silicone Oil
[0147] Samples containing about 2 mg/mL cetuximab in phosphate
buffer at pH 7 with 200 ppm excipient and 1% added silicone oil,
prepared in accordance with previous example, were analyzed for
insoluble particles by dynamic flow imaging with a FlowCam VS1
instrument (Fluid Imaging Technologies, Scarborough, Me.). The
FlowCam was equipped with a 10.times. objective lens and a 100
micron depth flow cell, and operated at a flow rate of 0.15 mL/min.
Measurements were made using a test fluid volume of 0.7 mL for each
sample. Particles were counted using the VisualSpreadsheet particle
analysis software provided with the FlowCam instrument, and
reported as total number of particles per milliliter. The results
of these tests are shown in Table 25 below, and selected results
are illustrated in FIG. 2.
TABLE-US-00025 TABLE 25 Excipient Excipient conc. Agitation FlowCam
Test No. added (ppm) (hrs) (Particles/mL) 4 PS80 200 40 92,633 5
PS80 200 40 51,252 6 PS80 200 40 52,254 10 PPG1000 200 40 90,594 11
PPG1000 200 40 31,638 12 PPG1000 200 40 60,551 16 None 0 40
3,852,450* 17 None 0 40 31,464,950* 18 None 0 40 22,273,530* 19
None 0 0 5,489 20 None 0 0 10,146 21 None 0 0 16,464 *Sample
diluted 10 fold with deionized water prior to analysis due to high
turbidity of sample.
EQUIVALENTS
[0148] While specific embodiments of the subject invention have
been disclosed herein, the above specification is illustrative and
not restrictive. While this invention has been particularly shown
and described with references to preferred embodiments thereof, it
will be understood by those skilled in the art that various changes
in form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims. Many
variations of the invention will become apparent to those of
skilled art upon review of this specification. Unless otherwise
indicated, all numbers expressing reaction conditions, quantities
of ingredients, and so forth, as used in this specification and the
claims are to be understood as being modified in all instances by
the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters set forth herein are approximations that
can vary depending upon the desired properties sought to be
obtained by the present invention.
* * * * *
References